Anti-GAPDH 抗体 [EPR16891] - Loading Control

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling GAPDH with ab181602 at 1/2000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Nucleus and cytoplasmic staining on lymphocyte of rat spleen is observed. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Intracellular flow cytometric analysis of 2% paraformaldehyde-fixed Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling GAPDH with ab181602 at 1/180 dilution (red) compared with a rabbit monoclonal IgG isotype control (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Immunocytochemistry/immunofluorescence staining of 4% paraformaldehyde fixed; 0.1% triton X 100 permeabilized HeLa (human cervix adenocarcinoma) cells labeling GAPDH with ab181602 at dilution of 1/500. The secondary antibody used was Alexa Fluor^® 488; goat anti-rabbit IgG (ab150077) at a dilution of 1/400. Nucleus was counter-stained with DAPI (blue). ab7291, a mouse anti-tubulin antibody (1/500) was used to stain tubulin along with ab150120 (AlexaFluor^®594 goat anti-mouse secondary, 1/500). The negative controls are shown in the bottom middle and right hand panels- for negative control 1 primary antibody (ab181602; 1/500) and secondary antibody (ab150120; 1/500) was used. For negative control 2 primary antibody (ab7291; 1/500) and secondary antibody (ab150077; 1/400) was used.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Immunohistochemical analysis of paraffin-embedded human transitional cell carcinoma of bladder tissue labeling GAPDH with ab181602 at 1/2000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasmic and nucleus staining on the tumor cells of transitional cell carcinoma of human bladder is observed. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Lab

Immunoprecipitation - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

STT3A was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney epithelial cell) whole cell lysate with ab320831 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab320831 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Immunoprecipitation, ab320831 was shown to bind specifically to STT3A. Target of interest was enriched at 60 kDa in wild-type HEK-293T cell lysates (lane 2) with no signal observed at this size in STT3A knockout cell line (lane 5) (lane 5, knockout cell line ab266320 / knockout cell lysate ab259164).
The bands above 60kDa are likely to be aggregation, and are absent in the KO samples. Unlike the Western Blot data, these samples have been boiled. ab181602 was used as a GAPDH loading control.

All lanes:

Immunoprecipitation - Anti-STT3A antibody [EPR29178-10] (<a href='/products/primary-antibodies/stt3a-antibody-epr29178-10-ab320831'>ab320831</a>) at 1/1000 dilution

Lane 1:

Parental HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 10 µg

Lane 2:

Parental HEK-293T (human embryonic kidney epithelial cell) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/products/primary-antibodies/stt3a-antibody-epr29178-10-ab320831'>ab320831</a> in Parental HEK-293T whole cell lysate

Lane 4:

STT3A KO HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 10 µg

Lane 5:

Immunoprecipitation - Human STT3A knockout HEK-293T cell line (<a href='/products/cell-lines/human-stt3a-knockout-hek-293t-cell-line-ab266320'>ab266320</a>)

Lane 6:

Rabbit monoclonal IgG (<a href='/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/products/primary-antibodies/stt3a-antibody-epr29178-10-ab320831'>ab320831</a> in STT3A KO HEK-293T whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 60 kDa

false

Exposure time: 84s

• IP

Supplier Data

Immunoprecipitation - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Dnmt3a was immunoprecipitated from 0.35 mg Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab323708 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab323708 at 1000 dilution.

Blocking and dilution buffer and concentration : 5% NFDM/TBST

All lanes:

Immunoprecipitation - Anti-Dnmt3a antibody [EPR29184-89] (<a href='/products/primary-antibodies/dnmt3a-antibody-epr29184-89-ab323708'>ab323708</a>) at 1/1000 dilution

Lane 1:

(Input) Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 10 µg

Lane 2:

<a href='/products/primary-antibodies/dnmt3a-antibody-epr29184-89-ab323708'>ab323708</a> at 1/30 IP in Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 10 µg

Lane 3:

Rabbit monoclonal IgG (<a href='/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/products/primary-antibodies/dnmt3a-antibody-epr29184-89-ab323708'>ab323708</a> in parental HeLa whole cell lysate at 10 µg

Lane 4:

(Input) DNMT3A knockout HeLa whole cell lysate at 10 µg

Lane 5:

<a href='/products/primary-antibodies/dnmt3a-antibody-epr29184-89-ab323708'>ab323708</a> at 1/30 IP in DNMT3A knockout HeLa whole cell lysate at 10 µg

Lane 6:

Rabbit monoclonal IgG (<a href='/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/products/primary-antibodies/dnmt3a-antibody-epr29184-89-ab323708'>ab323708</a> in DNMT3A knockout HeLa whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 130 kDa,36 kDa

false

Exposure time: 24s

• IP

Supplier Data

Immunoprecipitation - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

GAPDH was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell extract with ab181602 at 1/60 dilution. Western blot was performed from the immunoprecipitate using ab181602 at 1/5000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution. Lane 1 : HeLa whole cell extract.

Lane 2 : PBS instead of HeLa whole cell extract.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602)

Predicted band size: 36 kDa

Observed band size: 36 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling GAPDH with ab181602 at 1/2000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Nucleus and cytoplasmic staining on lymphocytes of mouse spleen is observed. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : MCF7, SK-BR-3 (PMID : 20563836), T-47D.

Samples are non-boiled as boiling may cause protein aggregation.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-PAR1/Thrombin Receptor antibody [EPR28399-240] (<a href='/products/primary-antibodies/par1-thrombin-receptor-antibody-epr28399-240-ab325686'>ab325686</a>) at 1/1000 dilution

Lane 1:

U-118 MG (human brain glioblastoma cell) whole cell lysate at 40 µg

Lane 2:

SK-N-SH (human neuroblastoma epithelial cell) whole cell lysate at 40 µg

Lane 3:

LN-229 (human brain glioblastoma epithelial cell) whole cell lysate at 40 µg

Lane 4:

NCI-H929 (human Bone B lymphocyte) whole cell lysate at 40 µg

Lane 5:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 40 µg

Lane 6:

T-47D (human ductal breast epithelial tumor epithelial cell) whole cell lysate at 40 µg

Lane 7:

SK-BR-3 (human breast adenocarcinoma epithelial cell) whole cell lysate at 40 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 47 kDa,70-100 kDa,36 kDa

false

Exposure time: 70s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

This blot was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-POLR3A antibody [EPR30480-88] (<a href='/products/primary-antibodies/polr3a-antibody-epr30480-88-ab325791'>ab325791</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

Hela transfected with siRNA specifically targeting POLR3A whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 155 kDa,36 kDa

true

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Samples are non-boiled as boiling may cause protein aggregation.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-IL-12RB1 antibody [EPR28354-118] (<a href='/products/primary-antibodies/il-12rb1-antibody-epr28354-118-ab325502'>ab325502</a>)

All lanes:

Human tonsil tissue lysate at 50 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 100 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Western blot : Rev-erbβ Antibody (D-8) absc-398252 staining at 1/1000 dilution, shown in green; Rabbit anti GAPDH (ab181602) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 70 kDa in Wild-type A549 cell lysates with no signal observed at this size in NR1D2 knockout A549 cell line (ab326030). To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse 800CW & Goat anti-Rabbit 680RD at 1/20,000 dilution.

All lanes:

Western blot at 1/1000 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

Western blot - Human NR1D2 knockout A549 cell line (<a href='/products/cell-lines/human-nr1d2-knockout-a549-cell-line-ab326030'>ab326030</a>) at 20 µg

Lane 3:

U-87 MG at 20 µg

Lane 4:

RKO at 20 µg

Secondary

All lanes:

Goat anti-Mouse 800CW & Goat anti-Rabbit 680RD at 1/20000 dilution

false

• WB

Supplier Data

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The identity of the bands higher than 100 kDa are unknown.

Lanes 1-2 are applied with Goat Anti-Rabbit IgG (HRP) with minimal cross reactivity with human IgG at 1/2000 and lanes 3-4 are applied with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-OMA1 antibody [EPR29198-78] (<a href='/products/primary-antibodies/oma1-antibody-epr29198-78-ab324586'>ab324586</a>) at 1/1000 dilution

Lanes 1 - 2:

Human heart tissue lysate at 20 µg

Lane 3:

HT-29 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

SK-MEL-28 (human malignant melanoma cell ) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : HeLa, 293T, MCF7.

The identity of the bands lower than 75 kDa are unknown.

Lanes 1-2 are applied with Goat Anti-Rabbit IgG (HRP) with minimal cross reactivity with human IgG at 1/2000 and lanes 3-8 are applied with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lanes 1-2 : 48 seconds; Lanes 3-4 : 59 seconds; Lanes 5-8 : 180 seconds.

All lanes:

Western blot - Anti-NFAT1 antibody [EPR30445-509] (ab325917) at 1/1000 dilution

Lane 1:

Human lymph node tissue lysate at 20 µg

Lane 2:

Human tonsil tissue lysate at 20 µg

Lane 3:

Jurkat (human t cell leukemia t lymphocyte from peripheral blood) whole cell lysate at 20 µg

Lane 4:

Ramos (human burkitt's lymphoma b lymphocyte) whole cell lysate at 20 µg

Lane 5:

Daudi (human burkitt's lymphoma lymphoblast) whole cell lysate at 20 µg

Lane 6:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 7:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 8:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

Lanes 1 - 2:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Lanes 3 - 8:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 100-140 kDa,36 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Performed under reducing conditions.

In Western blot, ab325539 was shown to bind specifically to BRD3. Target of interest was observed at 100 kDa in wild-type HEK-293 cell lysates (lane 1) with no signal observed at this size in BRD3 knockout cell line (lane 2) (lane 2, knockout cell line ab266793 / knockout cell lysate ab258335).

To minimize protein degradation, cells/tissues were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-BRD3 antibody [EPR30249-10] (<a href='/products/primary-antibodies/brd3-antibody-epr30249-10-ab325539'>ab325539</a>) at 1/1000 dilution

Lane 1:

Wild-type HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

Western blot - Human BRD3 knockout HEK-293T cell lysate (<a href='/products/cell-lysates/human-brd3-knockout-hek-293t-cell-lysate-ab258335'>ab258335</a>) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 100 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The expression of CXCL3 is upregulated in response to LPS treatment (PMID : 35789398).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lanes 1-2 : 180 senconds, Lanes 3-4 : 10 seconds

All lanes:

Western blot - Anti-GRO gamma antibody [EPR29736-526] (<a href='/products/primary-antibodies/gro-gamma-antibody-epr29736-526-ab325687'>ab325687</a>) at 1/1000 dilution

Lane 1:

Untreated THP-1 (human monocytic leukemia monocyte) whole cell lysate at 60 µg

Lane 2:

THP-1 treated first with 100ng/ml PMA for 24h, then replaced with 100ng/ml LPS for 4h, 1ug/ml BFA was then added for additional 3h, whole cell lysate at 60 µg

Lane 3:

Untreated HUVEC (human umbilical vein endothelial cell) whole cell lysate at 60 µg

Lane 4:

HUVEC treated with 0.5ug/ml LPS for 4h, 300ng/ml BFA was then added for additional 20h, whole cell lysate at 60 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 11 kDa,36 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : Ramos, Raji and MCF7.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Transglutaminase 2 antibody [RM1361] (<a href='/products/primary-antibodies/transglutaminase-2-antibody-rm1361-ab325401'>ab325401</a>) at 1/1000 dilution

Lane 1:

BxPC-3 (human pancreas adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HT-1080 (human fibrosarcoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

DU 145 (human prostate carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

MG-63 (human osteosarcoma fibroblast) whole cell lysate at 20 µg

Lane 5:

PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

786-O (human kidney epithelial cell) whole cell lysate at 20 µg

Lane 7:

Ramos (human Burkitt's lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 8:

Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 9:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 75 kDa,36 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-RAB43 antibody [EPR30492-530] (<a href='/products/primary-antibodies/rab43-antibody-epr30492-530-ab325751'>ab325751</a>) at 1/1000 dilution

Lane 1:

HepG2 (human hepatocellular carcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

HepG2 transfected with siRNA specifically targeting RAB43 whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 23 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Samples are non-boiled as boiling may cause protein aggregation.

Lane 2 of this blot was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-DMT1 antibody [EPR29592-29] (<a href='/products/primary-antibodies/dmt1-antibody-epr29592-29-ab324349'>ab324349</a>) at 1/1000 dilution

Lane 1:

Human pancreas cancer tissue lysate at 20 µg

Lane 2:

Human small intestine tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 75-100 kDa,36 kDa

true

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : liver (PMID : 24614806).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lanes 1-3 : 15 seconds, lanes 4-5 : 37 seconds, lanes 6-7 : 70 seconds

All lanes:

Western blot - Anti-BICD2 antibody [EPR30244-565] (<a href='/products/primary-antibodies/bicd2-antibody-epr30244-565-ab325398'>ab325398</a>) at 1/1000 dilution

Lane 1:

Mouse E14 brain tissue lysate at 20 µg

Lane 2:

Mouse skin tissue lysate at 20 µg

Lane 3:

Mouse liver tissue lysate at 20 µg

Lane 4:

Rat skin tissue lysate at 20 µg

Lane 5:

Rat liver tissue lysate at 20 µg

Lane 6:

Human cerebral cortex tissue lysate at 20 µg

Lane 7:

Human liver tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 100 kDa,36 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Performed under reducing conditions.

In Western blot, ab325401 was shown to bind specifically to TGM2. Target of interest was observed at 75 kDa in wild-type A549 cell lysates (lane 1) with no signal observed at this size in TGM2 knockout cell line (lane 2).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Transglutaminase 2 antibody [RM1361] (<a href='/products/primary-antibodies/transglutaminase-2-antibody-rm1361-ab325401'>ab325401</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 (human lung carcinoma epithelial cell) whole cell lysate at 40 µg

Lane 2:

TGM2 knockout A549 whole cell lysate at 40 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 75 kDa,36 kDa

false

Exposure time: 48s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000,000 dilution.

The full-length of E-cadherin is 120 kDa. The other bands are due to proteolytic cleavages in different Cadherin domains. (PMID : 14695147)

All lanes:

Western blot - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (<a href='/products/primary-antibodies/e-cadherin-antibody-ep700y-intercellular-junction-marker-ab40772'>ab40772</a>) at 1/1000 dilution

All lanes:

U266B1 (human multiple myeloma B lymphocyte) whole cell lysate, at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 120 kDa

Observed band size: 80-120 kDa,36 kDa

false

Exposure time: 7s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : testis, liver (PMID : 37821487).

Samples are non-boiled as boiling may cause protein aggregation.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-NOX5 antibody [EPR29814-35] (<a href='/products/primary-antibodies/nox5-antibody-epr29814-35-ab325647'>ab325647</a>) at 1/1000 dilution

Lane 1:

Human spleen tissue lysate at 20 µg

Lane 2:

Human testis tissue lysate at 20 µg

Lane 3:

Human liver tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 75 kDa,36 kDa

false

Exposure time: 1s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Samples are non-boiled as boiling may cause protein aggregation.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-NOX5 antibody [EPR29814-35] (<a href='/products/primary-antibodies/nox5-antibody-epr29814-35-ab325647'>ab325647</a>) at 1/1000 dilution

Lane 1:

HT-29 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

T-47D (human ductal breast epithelial tumor epithelial cell) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 75 kDa,36 kDa

false

Exposure time: 6s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In lanes 1-2 on the right, the lysates was stored at -80degC prior to Western Blotting. The bands beneath the target band (100 kDa) are likely to be degradation products. In lanes 1-2 on the left, to minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-BRD3 antibody [EPR30249-10] (<a href='/products/primary-antibodies/brd3-antibody-epr30249-10-ab325539'>ab325539</a>) at 1/1000 dilution

Lane 1:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 2:

MEF (mouse embryo fibroblast) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 100 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : skeletal muscle.

The weak band observed at ~35KDa may represent the precursor form of GILT (PMID : 39273610).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-GILT antibody [EPR30714-572] (<a href='/products/primary-antibodies/gilt-antibody-epr30714-572-ab325920'>ab325920</a>) at 1/1000 dilution

Lane 1:

Human liver tissue lysate at 20 µg

Lane 2:

Human breast cancer tissue lysate at 20 µg

Lane 3:

Human lung tissue lysate at 20 µg

Lane 4:

Human spleen tissue lysate at 20 µg

Lane 5:

Human lymph node tissue lysate at 20 µg

Lane 6:

Human tonsil tissue lysate at 50 µg

Lane 7:

Human lung tissue lysate at 50 µg

Lane 8:

Human skeletal muscle tissue lysate at 50 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 28 kDa,35 kDa,36 kDa

false

Exposure time: 3s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Negative control : testis, lung, pancreas

Lanes 1-4 are applied with Goat Anti-Rabbit IgG (HRP) with minimal cross reactivity with human IgG at 1/2000 and lanes 5-8 are applied with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-TDO2/TDO antibody [EPR29352-720] (<a href='/products/primary-antibodies/tdo2-tdo-antibody-epr29352-720-ab325323'>ab325323</a>) at 1/1000 dilution

Lane 1:

Human liver tissue lysate at 40 µg

Lane 2:

Human testis tissue lysate at 40 µg

Lane 3:

Human lung tissue lysate at 40 µg

Lane 4:

Human pancreas tissue lysate at 40 µg

Lane 5:

Mouse liver tissue lysate at 40 µg

Lane 6:

Mouse testis tissue lysate at 40 µg

Lane 7:

Mouse lung tissue lysate at 40 µg

Lane 8:

Mouse pancreas tissue lysate at 40 µg

Secondary

Lanes 1 - 4:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Lanes 5 - 8:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 43 kDa,36 kDa

false

Exposure time: 26s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Western blot : Mouse Monoclonal[4C6] to DMT1 ab55735 staining at 1/1000 dilution, shown in green; Rabbit anti GAPDH (ab181602) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 80 kDa in Wild-type A549 cell lysates with no signal observed at this size in SLC11A2 knockout A549 cell line (ab326025). To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse 800CW & Goat anti-Rabbit 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-DMT1 antibody [4C6] (<a href='/products/primary-antibodies/dmt1-antibody-4c6-ab55735'>ab55735</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

Western blot - Human SLC11A2 knockout A549 cell line (<a href='/products/cell-lines/human-slc11a2-knockout-a549-cell-line-ab326025'>ab326025</a>) at 20 µg

Secondary

All lanes:

Goat anti-Mouse 800CW & Goat anti-Rabbit 680RD at 1/20000 dilution

Predicted band size: 62 kDa

Observed band size: 80 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : THP-1.

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

The identity of the lower MW band at approximately 60 kDa is unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-INPP5F antibody [MJF-D29759-51] (<a href='/products/primary-antibodies/inpp5f-antibody-mjf-d29759-51-ab325711'>ab325711</a>) at 1/1000 dilution

Lane 1:

SK-MEL-28 (human malignant melanoma cell) whole cell lysate at 20 µg

Lane 2:

THP-1 (human monocytic leukemia monocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 140 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

In Western blot, Anti-GSDMD antibody [EPR20859] (ab219800) 1 : 1,000 dilution (0.582 ug/ml) and Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) 1 : 1,000,000 dilution.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-GSDMD antibody [EPR20859] (<a href='/products/primary-antibodies/gsdmd-antibody-epr20859-ab219800'>ab219800</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

Jurkat (human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 53 kDa

Observed band size: 53 kDa,37 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : HeLa.

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

The identity of the lower MW band at approximately 60 kDa is unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-INPP5F antibody [MJF-D29759-51] (<a href='/products/primary-antibodies/inpp5f-antibody-mjf-d29759-51-ab325711'>ab325711</a>) at 1/1000 dilution

Lane 1:

A375 (human malignant melanoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 140 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-POLR3A antibody [EPR30480-88] (<a href='/products/primary-antibodies/polr3a-antibody-epr30480-88-ab325791'>ab325791</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

SK-N-SH (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 7:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 8:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 155 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Performed under reducing conditions.

In Western blot, ab325397 was shown to bind specifically to UFL1. Target of interest was observed at 40, 80 kDa in wild-type HEK-293 cell lysates (lane 3) with no signal observed at this size in UFL1 knockout HEK-293 cell line (lane 4) (lane 4, knockout cell line ab266500 / knockout cell lysate ab258748).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-UFL1 antibody [EPR29684-84] (<a href='/products/primary-antibodies/ufl1-antibody-epr29684-84-ab325397'>ab325397</a>) at 1/1000 dilution

Lane 1:

EL4 (mouse lymphoma T lymphocyte) whole cell lysate at 50 µg

Lane 2:

B16-F10 (mouse skin melanoma cell) whole cell lysate at 50 µg

Lane 3:

Wild-type HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 50 µg

Lane 4:

Western blot - Human UFL1 knockout HEK-293T cell lysate (<a href='/products/cell-lysates/human-ufl1-knockout-hek-293t-cell-lysate-ab258748'>ab258748</a>) at 50 µg

Lane 5:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 50 µg

Lane 6:

HaCaT (human skin keratinocyte) whole cell lysate at 50 µg

Lane 7:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 50 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 40 kDa,80 kDa,36 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : kidney, liver(PMID : 25760323).

The samples are unboiled.

The band pattern observed is consistent with what has been described in the literature (PMID : 34833992).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Cannabinoid Receptor I antibody [EPR30843-34] (<a href='/products/primary-antibodies/cannabinoid-receptor-i-antibody-epr30843-34-ab325797'>ab325797</a>) at 1/1000 dilution

Lane 1:

Human cerebellum tissue lysate at 40 µg

Lane 2:

Human kidney tissue lysate at 40 µg

Lane 3:

Mouse brain tissue lysate at 40 µg

Lane 4:

Mouse cerebellum tissue lysate at 40 µg

Lane 5:

Mouse kidney tissue lysate at 40 µg

Lane 6:

Mouse liver tissue lysate at 40 µg

Lane 7:

Rat brain tissue lysate at 40 µg

Lane 8:

Rat cerebellum tissue lysate at 40 µg

Lane 9:

Rat kidney tissue lysate at 40 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 37 kDa,50 kDa,36 kDa

false

Exposure time: 6s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Performed under reducing conditions.

In Western blot ab325939 was shown to bind specifically to Dishevelled 2. Target of interest was observed at 95 kDa in wild-type A549 cell lysates (lane 3) with no signal observed at this size in Dishevelled 2 knockout cell line (lane 4) (knockout cell line ab277903 / knockout cell lysate ab288317).

Low expression : liver lung.

In Western blot Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lanes 1-4 : 26 seconds; Lane 5 : 6 seconds; Lane 6 : 26 seconds.

All lanes:

Western blot - Anti-Dishevelled 2 antibody [EPR30298-74] (<a href='/products/primary-antibodies/dishevelled-2-antibody-epr30298-74-ab325939'>ab325939</a>) at 1/1000 dilution

Lane 1:

Human ovary tissue lysate at 20 µg

Lane 2:

Human testis tissue lysate at 20 µg

Lane 3:

Human liver tissue lysate at 20 µg

Lane 4:

Human lung tissue lysate at 20 µg

Lane 5:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

Dishevelled 2 knockout A549 whole cell lysate at 20 µg

Lane 7:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 8:

NCI-H1299 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 95 kDa,36 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

To minimize protein degradation, lysates were made fresh.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lanes 1-2 : 180 seconds, lanes 3-4 : 180 seconds

All lanes:

Western blot - Anti-Zfp281/ZNF281 antibody [EPR30593-518] (<a href='/products/primary-antibodies/zfp281-znf281-antibody-epr30593-518-ab325790'>ab325790</a>) at 1/1000 dilution

Lane 1:

U-2 OS (human bone osteosarcoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

U-2 OS transfected with siRNA specifically targeting ZNF281 whole cell lysate at 20 µg

Lane 3:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 100 kDa,36 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Performed under reducing conditions.

In Western blot, ab324680 was shown to bind specifically to NUR77. Target of interest was observed at 65-75 kDa in wild-type HeLa cell lysates (lane 1) with no signal observed at this size in NR4A1 knockout cell line (lane 2) (lane 2, knockout cell line ab264021 / knockout cell lysate ab264509).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-NUR77 antibody [EPR29846-533] (<a href='/products/primary-antibodies/nur77-antibody-epr29846-533-ab324680'>ab324680</a>) at 1/1000 dilution

Lane 1:

Wild type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 50 µg

Lane 2:

NR4A1 knockout HeLa whole cell lysate at 50 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 65-75 kDa,36 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Negative control : HeLa.

This blot was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-TDO2/TDO antibody [EPR29352-720] (<a href='/products/primary-antibodies/tdo2-tdo-antibody-epr29352-720-ab325323'>ab325323</a>) at 1/1000 dilution

Lane 1:

HDLM-2 (human Hodgkin lymphoma cell) whole cell lysate at 48 µg

Lane 2:

LoVo (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 48 µg

Lane 3:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 48 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/2000 dilution

Observed band size: 43 kDa,36 kDa

true

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Performed under reducing conditions.

In Western blot, ab324349 was shown to bind specifically to DMT1/SLC11A2 . Target of interest was observed at 75-100 kDa in wild-type HeLa cell lysates (lane 1) with no signal observed at this size in DMT1/SLC11A2 knockout cell line (lane 2).

Samples are non-boiled as boiling may cause protein aggregation.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-DMT1 antibody [EPR29592-29] (<a href='/products/primary-antibodies/dmt1-antibody-epr29592-29-ab324349'>ab324349</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

DMT1/SLC11A2 knockout HeLa whole cell lysate at 20 µg

Lane 3:

SK-MEL-2 (human skin malignant melanoma cell) whole cell lysate at 20 µg

Lane 4:

SK-MEL-28 (human malignant melanoma cell) whole cell lysate at 20 µg

Lane 5:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 75-100 kDa,36 kDa

false

Exposure time: 92s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In lanes 1 and 4, the lysates were stored at -80degC prior to Western Blotting. The bands beneath the target band (95 kDa) are likely to be degradation products. In lanes 2-3, to minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Dishevelled 2 antibody [EPR30298-74] (<a href='/products/primary-antibodies/dishevelled-2-antibody-epr30298-74-ab325939'>ab325939</a>) at 1/1000 dilution

Lane 1:

HEK-293 (human embryonic kidney epithelial cell) frozen whole cell lysate at 20 µg

Lane 2:

HEK-293 (human embryonic kidney epithelial cell) fresh whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) fresh whole cell lysate at 20 µg

Lane 4:

NIH/3T3 (mouse embryonic fibroblast) frozen whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 95 kDa,36 kDa

false

Exposure time: 59s

• WB

Supplier Data

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-COX IV antibody [EPR9442 (ABC)] - Mitochondrial Loading Control (ab202554) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-OMA1 antibody [EPR29198-78] (<a href='/products/primary-antibodies/oma1-antibody-epr29198-78-ab324586'>ab324586</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) mitochondria fraction at 20 µg

Lane 2:

HeLa non-mitochondrial fraction at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 36 kDa,16 kDa

false

Exposure time: 180s