Anti-BCKDHA (phospho S293) 抗体 [EPR27489-76] (ab302504)

All lanes : Anti-BCKDHA (phospho S293) antibody [EPR27489-76] (ab302504) at 1/1000 dilution

Lane 1 : Wild-type HEK293T (human embryonic kidney epithelial cell), whole cell lysate (untreated membrane)
Lane 2 : BCKDHA knockout HEK293T whole cell lysate (untreated membrane)
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate (untreated membrane)
Lane 4 : C6 (rat glial tumor glial cell), whole cell lysate (untreated membrane)
Lane 5 : mIMCD3 (mouse inner medullary collecting duct epithelial cell), whole cell lysate (untreated membrane)
Lane 6 : Wild-type HEK293T (human embryonic kidney epithelial cell), whole cell lysate (phosphatase treated membrane)
Lane 7 : BCKDHA knockout HEK293T whole cell lysate (phosphatase treated membrane)
Lane 8 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate (phosphatase treated membrane)
Lane 9 : C6 (rat glial tumor glial cell), whole cell lysate (phosphatase treated membrane)
Lane 10 : mIMCD3 (mouse inner medullary collecting duct epithelial cell), whole cell lysate (phosphatase treated membrane)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (ab216773) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (ab216776) at 1/10000 dilution

Predicted band size: 50 kDa
Observed band size: 49 kDa why is the actual band size different from the predicted?

Blocking / Diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.

Samples were run on a Bis-Tris gel.
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
False colour image of Western blot: Anti- BCKDHA (phospho S293) antibody (ab302504) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red.
In Western blot, ab302504 was shown to bind specifically to BCKDHA (phospho S293) . A band was observed at 49 kDa in wild-type HEK293T cell lysates with no signal observed at this size in BCKDHA knockout cell line ab266439 (knockout cell lysate ab258324). To generate this image, wild-type and BCKDHA knockout HEK293T cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in in Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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.

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling BCKDHA (phospho S293) with ab302504 at 1/500 dilution (0.948 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Mitochondrial staining on human colon without alkaline phosphatase treatment (Panel A). No signal was detected when tissues were treated with alkaline phosphatase (Panel B). The section was incubated with ab302504 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used.

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling BCKDHA (phospho S293) with ab302504 at 1/500 dilution (0.948 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Mitochondrial staining on human colon cancer without alkaline phosphatase treatment (Panel A). No signal was detected when tissues were treated with alkaline phosphatase (Panel B) (PMID:32238881). The section was incubated with ab302504 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used.

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling BCKDHA (phospho S293) with ab302504 at 1/500 dilution (0.948 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Mitochondrial staining on mouse kidney without alkaline phosphatase treatment (Panel A). No signal was detected when tissues were treated with alkaline phosphatase (Panel B). The section was incubated with ab302504 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used.

Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling BCKDHA (phospho S293) with ab302504 at 1/500 dilution (0.948 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Mitochondrial staining on mouse liver cancer without alkaline phosphatase treatment (Panel A). No signal was detected when tissues were treated with alkaline phosphatase (Panel B). The section was incubated with ab302504 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used.

Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling BCKDHA (phospho S293) with ab302504 at 1/500 dilution (0.948 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Mitochondrial staining on rat kidney without alkaline phosphatase treatment (Panel A). No signal was detected when tissues were treated with alkaline phosphatase (Panel B). The section was incubated with ab302504 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used.

Immunohistochemical analysis of paraffin-embedded HEK-293T cell pellets labeling BCKDHA (phospho S293) with ab302504 at 1/500 dilution (0.948 µg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Mitochondrial staining on HEK-293T without alkaline phosphatase treatment (Panel A). No signal was detected when cells were treated with alkaline phosphatase (Panel B). No staining on BCKDHA KO HEK-293T cell pellets (Panel C). The section was incubated with ab302504 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used.

BCKDHA (phospho S293) was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate 10 µg with AB302504 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab302504 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate 10 µg (Inset)

Lane 2: ab302504 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab302504 in Hela whole cell lysate

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

Exposure time: 180 seconds

 

BCKDHA (phospho S293) was immunoprecipitated from 0.35 mg mIMCD3 (mouse inner medullary collecting duct epithelial cell), whole cell lysate 10 µg with ab302504 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab302504 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: mIMCD3 (mouse inner medullary collecting duct epithelial cell), whole cell lysate 10 µg (Inset)

Lane 2: ab302504 IP in mIMCD3 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab302504 in mIMCD3 whole cell lysate

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

Exposure time: 180 seconds