All lanes : Anti-TIA1 antibody [EPR22999-80] (ab263945) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : TIA1 knockout HAP1 whole cell lysate
Lane 3 : Jurkat (human T cell leukemia T lymphocyte), whole cell lysate
Lane 4 : HuT-78 (human Sezary syndrome cutaneous T lymphocyte), whole cell lysate
Lane 5 : MOLT-4 (human lymphoblastic leukemia T lymphoblast), whole cell lysate
Lane 6 : K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate
Lane 7 : Human tonsil tissue 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: 43 kDa
Observed band size: 42,43 kDa why is the actual band size different from the predicted?

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

The molecular weight observed is consistent with what has been described in the literature (PMID:30533021).

ab263945 was shown to specifically react with TIA1 in wild-type HAP1 cells as signal was lost in TIA1 knockout cells. Wild-type and TIA1 knockout samples were subjected to SDS-PAGE. ab263945 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD^® ChemiDoc^™ MP instrument using the ECL technique. 

Exposure time: Lanes 1-3: 103 seconds Lane 4: 26 seconds Lanes 5-6: 103 seconds Lane 7: 3 minutes.

Immunohistochemical analysis of paraffin-embedded Human gastric cancer tissue labeling TIA1 with ab263945 at 1/4000 dilution (0.15ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in tumor-infiltrating T-lymphocytes of human gastric cancer (PMID: 10658910) is observed.

The section was incubated with ab256492 for 15 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.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized TIA1 knockout HAP1 (TIA1 knockout human chronic myelogenous leukemia near-haploid cell line, (Left) / WT HAP1 (Right) cells labelling TIA1 with ab263945 at 1/600 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

TIA1 was immunoprecipitated from 0.35 mg HuT-78 (human Sezary syndrome cutaneous T lymphocyte) whole cell lysate with ab263945 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab263945 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: HuT-78 (human Sezary syndrome cutaneous T lymphocyte) whole cell lysate 10ug

Lane 2: ab263945 IP in HuT-78 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab263945 in HuT-78 whole cell lysate

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

Exposure time: 3 seconds.

Truncated form (~16kDa) of TIA1 was also immunoprecipitated. (PMID: 25224594).

 

TIA1 was immunoprecipitated from 0.35 mg Jurkat (human T cell leukemia T lymphocyte) whole cell lysate 10ug with ab263945 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab263945 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: Jurkat (human T cell leukemia T lymphocyte) whole cell lysate 10ug

Lane 2: ab263945 IP in Jurkat whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab263945 in Jurkat whole cell lysate

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

Exposure time: 10 seconds.

Truncated form (~16kDa) of TIA1 was also immunoprecipitated. (PMID: 25224594).

 

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized Jurkat (human T cell leukemia T lymphocyte) cells labelling TIA1 with ab263945 at 1/600 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded Human classical hodgkin lymphoma tissue labeling TIA1 with ab263945 at 1/4000 dilution (0.15ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in T-lymphocytes of human classical hodgkin lymphoma (PMID: 19096012) is observed.

The section was incubated with ab256492 for 15 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.

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling TIA1 with ab263945 at 1/4000 dilution (0.15ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in T-lymphocytes of human spleen (PMID: 23510456) is observed.

The section was incubated with ab256492 for 15 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.