Anti-PAK1 抗体 [EPR26067-45] (BSA and Azide free) (ab302507)

All lanes : Anti-PAK1 antibody [EPR26067-45] (ab302506) at 1/1000 dilution

Lane 1 : Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate
Lane 2 : PAK1 knockout HAP1 whole cell lysate
Lane 3 : SH-SY5Y (human neuroblastoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 61 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?


Exposure time: 37 seconds

This data was developed using ab302506, the same antibody clone in a different buffer formulation.

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

Performed under reducing conditions.

In Western blot, ab302506 was shown to bind specifically to PAK1. A band was observed at 68 kDa in wild-type HAP1 cell lysates whereas no signal was observed at this size in PAK1 knockout cell lysates.

All lanes : Anti-PAK1 antibody [EPR26067-45] (ab302506) at 1/1000 dilution

Lane 1 : HEK-293 (human embryonic kidney epithelial cell), whole cell lysate
Lane 2 : SK-OV-3 (human ovarian cancer epithelial cell), whole cell lysate
Lane 3 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate
Lane 4 : C6 (rat glial tumor glial cell), whole cell lysate
Lane 5 : Mouse brain tissue lysate
Lane 6 : Rat brain tissue lysate
Lane 7 : Human kidney tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1-3 & 5-7 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lane 4 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

Predicted band size: 61 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?

This data was developed using ab302506, the same antibody clone in a different buffer formulation.

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

Exposure time:

Lane1-4: 15 seconds
Lane 5-6: 3.25 seconds
Lane 7: 37 seconds

This data was developed using ab302506, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human gastric cancer tissue labeling PAK1 with ab302506 at 1/500 dilution (0.115 µg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection kit). Cytoplasmic staining on human gastric cancer tissue and no staining on adjacent tissue. The section was incubated with ab302506 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 Leica DS9800 (BOND™ Polymer Refine Detection kit).

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

This data was developed using ab302506, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human ovarian cancer tissue labeling PAK1 with ab302506 at 1/500 dilution (0.115 µg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection kit). Cytoplasmic staining on human ovarian cancer. The section was incubated with ab302506 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 Leica DS9800 (BOND™ Polymer Refine Detection kit).

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

This data was developed using ab302506, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded mouse pancreas cancer tissue labeling PAK1 with ab302506 at 1/500 dilution (0.115 µg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection kit). Cytoplasmic staining on mouse pancreas cancer. The section was incubated with ab302506 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 Leica DS9800 (BOND™ Polymer Refine Detection kit).

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

This data was developed using ab302506, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labeling PAK1 with ab302506 at 1/2000 dilution (0.287 µg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection kit). Cytoplasmic staining on rat cerebrum. The section was incubated with ab302506 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 Leica DS9800 (BOND™ Polymer Refine Detection kit).

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

This data was developed using ab302506, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling PAK1 with ab302506 at 1/2000 dilution (0.287 µg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection kit). Cytoplasmic staining on mouse cerebrum. The section was incubated with ab302506 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 Leica DS9800 (BOND™ Polymer Refine Detection kit).

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

This data was developed using ab302506, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded knockout and Wild-type HAP1 cell pellets. ab302506 labeling PAK1 at 1/500 dilution (1.146 µg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection kit). Cytoplasmic staining on (A) wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) cell pellet and no staining on (B) PAK1 knockout HAP1 cell pellet. The section was incubated with ab302506 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 Leica DS9800 (BOND™ Polymer Refine Detection kit).

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

This data was developed using ab302506, the same antibody clone in a different buffer formulation.

PAK1 was immunoprecipitated from 0.35 mg SH-SY5Y (human neuroblastoma epithelial cell), whole cell lysate 10 µg with ab302506 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab302506 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: SH-SY5Y (human neuroblastoma epithelial cell), whole cell lysate 10 µg (Inset)

Lane 2: ab302506 IP in SH-SY5Y whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab302506 in mouse eyeball tissue lysate.

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

Exposure time: 24 seconds

This data was developed using ab302506, the same antibody clone in a different buffer formulation.

Dot blot analysis of PAK1 using ab302506 at 1:1000 dilution (0.573 µg/mL) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1:100,000 dilution.

Lane 1: Human PAK1 peptide

Lane 2: Human PAK3 peptide corresponding to the region homologous to human PAK1 peptide

Exposure time: 3 minutes

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