All lanes : Anti-GSTK1 antibody [EPR1939] (ab134173) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : GSTK1 knockout HEK293T cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : Daudi 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: 25 kDa
Observed band size: 27 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab134173 observed at 27 kDa. Red - loading control ab8245 observed at 36 kDa. 

 ab134173 Anti-GSTK1 antibody [EPR1939] was shown to specifically react with GSTK1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266112 (knockout cell lysate ab257461) was used.  Wild-type and GSTK1 knockout samples were subjected to SDS-PAGE.  ab134173 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours 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.

All lanes : Anti-GSTK1 antibody [EPR1939] (ab134173) at 1/1000 dilution (Purified)

Lane 1 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 25 kDa
Observed band size: 25 kDa

Blocking/Diluting buffer: 5% NFDM/TBST

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue sections labeling GSTK1 with purified ab134173 at 1/300 dilution (0.99 µg/mL). Heat mediated antigen retrieval using Bond™™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunocytochemistry/ Immunofluorescence analysis of 293T (Human embryonic kidney epithelial cell) cells labeling GSTK1 with Purified ab134173 at 1/50 dilution (5.92 µg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Purified ab134173 at 1/30 dilution (2ug) immunoprecipitating GSTK1 in HeLa whole cell lysate.
Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate (10µg)
Lane 2 (+): ab134173 + HeLa whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab134173 in HeLa whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 25 kDa

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling GSTK1 with Purified ab134173 at 1/30 dilution (10 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).