ab140595 staining TIA1 in wild-type Hap1 cells (top panel) and TIA1 knockout Hap1 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab140595 at 1/250 dilution and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

All lanes : Anti-TIA1 antibody [EPR9304] (ab140595) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : TIAL1 knockout HeLa cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : K-562 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 43 kDa
Observed band size: 42-43 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab140595 observed at 42-43 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab140595 Anti-TIA1 antibody [EPR9304] was shown to specifically react with TIA1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265793 (knockout lysate ab257743) was used. Wild-type and TIA1 knockout samples were subjected to SDS-PAGE. ab140595 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were iincubated overnight at 4°C at 1 in 1000  dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human spleen tissue labelling TIA1 with purified ab140595 at 1/1000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunocytochemistry/Immunofluorescence analysis of HuT-78 cells labelling TIA1 with purified ab140595 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

Lane 1: Wild-type HAP1 cell lysate (40 µg)
Lane 2: TIA1 knockout HAP1 cell lysate (40 µg)
Lane 3: Jurkat cell lysate (40 µg)
Lane 4: K562 cell lysate (40 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab140595 observed at 43 kDa. Red - loading control, ab18058, observed at 124 kDa.

ab140595 was shown to specifically react with TIA1  when TIA1  knockout samples were used. Wild-type and TIA1  knockout samples were subjected to SDS-PAGE. Ab140595 and ab18058 (loading control to Vinculin) were diluted at 1/1000 and 1/10000 dilution respectively and incubated overnight at 4C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

Anti-TIA1 antibody [EPR9304] (ab140595) at 1/5000 dilution (purified) + NIH/3T3 whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

Predicted band size: 43 kDa
Observed band size: 42-43 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-TIA1 antibody [EPR9304] (ab140595) at 1/5000 dilution (purified)

Lane 1 : Jurkat whole cell lysate
Lane 2 : K562 whole cell lysate
Lane 3 : HUT-78 whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

Predicted band size: 43 kDa
Observed band size: 42-43 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-TIA1 antibody [EPR9304] (ab140595) (unpurified)

Lane 1 : HuT-78 cell lysate
Lane 2 : Jurkat cell lysate
Lane 3 : Molt-4 cell lysate
Lane 4 : K562 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 43 kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human spleen tissue labelling TIA1 with unpurified ab140595 at a dilution of 1/100.

ab140595 (purified) at 1/40 immunoprecipitating TIA1 in HuT-78 whole cell lysate.

Lane 1 (input): HuT-78 whole cell lysate (10µg)

Lane 2 (+): ab140595 + HuT-78 whole cell lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab140595 in HuT-78 whole cell lysate.

For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.