Anti-YB1 抗体

• WB

Ap

Western blot - Anti-YB1 antibody (AB12148)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab12148 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

All lanes:

Western blot - Anti-YB1 antibody (ab12148) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 10 µg

Lane 3:

Jurkat (Human) Whole Cell Lysate at 10 µg

Lane 4:

Western blot - T-47D whole cell lysate (<a href='/products/cell-lysates/t-47d-whole-cell-lysate-ab14899'>ab14899</a>) at 10 µg

Lane 5:

MDA-MB-231 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/50000 dilution

Predicted band size: 36 kDa

Observed band size: 100 kDa,50 kDa

true

Exposure time: 4min

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-YB1 antibody (AB12148)

ICC/IF image of ab12148 stained human HeLa cells. The cells were PFA fixed (3.7% PFA, 5 min) and incubated with the antibody (ab12148, 1μg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT™ FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor^® 594 WGA was used to label plasma membranes (red).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-YB1 antibody (AB12148)

ICC/IF image of ab12148 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab12148, 1μg/ml) overnight at +4°C. The secondary antibody (green) was DyLight^® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43μM.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-YB1 antibody (AB12148)

ICC/IF image of ab12148 stained human HeLa cells. The cells were PFA fixed (3.7% PFA, 5 min) and incubated with the antibody (ab12148, 1μg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT™ FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor^® 594 WGA was used to label plasma membranes (red).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-YB1 antibody (AB12148)

Immunohistochemical analysis of paraffin-embedded Human breast tissue labeling YB1 with ab320005 at 1/5000 (0.098 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human breast in (A) ab320005 and (B) ab12148. The image on (A) ab320005 is applied with Anti-YBX1 antibody at 1/5000 dilution. The image on (B) ab12148 is applied with Anti-YBX1 antibody at 1/200 dilution. The section was incubated with ab320005 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• WB

Unknown

Western blot - Anti-YB1 antibody (AB12148)

Lanes 1 to 10 : ab12148 - Rabbit polyclonal to YB1 (1.4μg/ml)

Lanes 1 to 10 : 20μg of cell lysate per lane
Lanes 6 to 10 : blocking peptide used at 1μg/ml

Lane 1 : HeLa Nuclear Extract
Lane 2 : HeLa Whole Cell Lysate
Lane 3 : MCF-7 Cell Lysate
Lane 4 : Jurkat Whole Cell Lysate
Lane 5 : HEK293 Whole Cell Lysate
Lane 6 : HeLa Nuclear Extract + YB1 blocking peptide (ab12411)
Lane 7 : HeLa Whole Cell Lysate + YB1 blocking peptide (ab12411)
Lane 8 : MCF-7 Cell Lysate + YB1 blocking peptide (ab12411)
Lane 9 : Jurkat Whole Cell Lysate + YB1 blocking peptide (ab12411)
Lane 10 : HEK293 Whole Cell Lysate + YB1 blocking peptide (ab12411)

Expected MW : 36 kDa

A band of approx 36 kDa was partially blocked in several cell lines using a YB1 blo

All lanes:

Western blot - Anti-YB1 antibody (ab12148) at 1.4 µg/mL

Lanes 1 and 6:

HeLa Nuclear lysate at 20 µg

Lanes 2 and 7:

HeLa Whole cell lysate at 20 µg

Lanes 3 and 8:

MCF-7 cell lysate at 20 µg

Lanes 4 and 9:

Jurkat whole cell lysate at 20 µg

Lane 5:

HEK293 Whole cell lysate at 20 µg

Lane 10:

HEK293 whole cell lysate at 20 µg

Predicted band size: 36 kDa

Observed band size: 36 kDa,50 kDa

false

• WB

Supplier Data

Western blot - Anti-YB1 antibody (AB12148)

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

The molecular weight observed is consistent with what has been described in the literature (PMID : 27168020, PMID : 24107631).

This blot of polyclonal ab12148 was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

The identity of the higher MW band at approximately 15 kDa and between 50 kDa and 150 kDa (Lane 2, 4, 6) are unknown.

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

All lanes:

Western blot - Anti-YB1 antibody [RM1188] (<a href='/products/primary-antibodies/yb1-antibody-rm1188-ab320005'>ab320005</a>) at 1/5000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 10 µg

Lane 2:

SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 10 µg

Lane 3:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 10 µg

Lane 4:

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

Lane 5:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 10 µg

Lane 6:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 10 µg

Lane 7:

C6 (rat glial tumor glial cell) whole cell lysate at 10 µg

Lane 8:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate at 10 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: 50 kDa,36 kDa

false

• WB

Project

Western blot - Anti-YB1 antibody (AB12148)

YB1 has a predicted band size of 36kDa based on its primary sequence (SwissProt). According to Evdolimova (1995) YB1 migrates by SDS-PAGE at 50kDa, which may be due to post-translational modification

All lanes:

Western blot - Anti-YB1 antibody (ab12148) at 1 µg/mL

All lanes:

HEK293 Whole Cell Lysate Transiently Overexpressing YB1 at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 36 kDa

Observed band size: 50 kDa

false