Recombinant Anti-Phospholipase C gamma 1/PLC-gamma-1 antibody [10-PLC-gamma] (ab302940)

All lanes : Anti-Phospholipase C gamma 1/PLC-gamma-1 antibody [10-PLC-gamma] (ab302940) at 1/1000 dilution

Lane 1 : Wild type HEK-293T (human embryonic kidney epithelial cell), whole cell lysate
Lane 2 : Phospholipase C gamma 1/PLC-gamma-1 knockout HEK-293T whole cell lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 4 : HepG2 (human hepatocellular carcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 148 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?
Additional bands at: 36 kDa (possible Loading Control)

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

False colour image of Western blot: Anti-Phospholipase C gamma 1/PLC-gamma-1 antibody [10/PLCgamma] (ab302940 staining at 1/1000 dilution, shown in green; Rabbit anti-GAPDH antibody [16891] (ab181602) loading control staining at 1/20000 dilution, shown in red.

In Western blot, ab302940 was shown to bind specifically to Phospholipase C gamma 1/PLC-gamma-1. A band was observed at 150kDa in wild-type HEK-293 cell lysates with no signal observed at this size in the Phospholipase C gamma 1/PLC-gamma-1 knockout cell line. To generate this image, wild-type and Phospholipase C gamma 1/PLC-gamma-1 knockout HEK-293 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a PVDF-FL membrane. Membranes were blocked in Odyssey diluted in 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. Secondary antibodies used were Goat Anti-Mouse IgG H&L (IRDye^® 800CW) (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) (ab216777) at 1/10000 dilution.

Performed under reducing conditions.

All lanes : Anti-Phospholipase C gamma 1/PLC-gamma-1 antibody [10-PLC-gamma] (ab302940) at 1/1000 dilution

Lane 1 : C6 (rat glial tumor glial cell), whole cell lysate
Lane 2 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate
Lane 3 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate
Lane 4 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate
Lane 5 : Mouse brain tissue lysate
Lane 6 : Mouse heart tissue lysate
Lane 7 : Mouse kidney tissue lysate
Lane 8 : Mouse spleen tissue lysate
Lane 9 : Rat brain tissue lysate
Lane 10 : Rat heart tissue lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 148 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?

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

Exposure time: Lanes 1-8: 72 seconds, Lanes 9-10: 59 seconds. 

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized MCF7 (Human breast adenocarcinoma cell line) cells labeling Phospholipase Cγ1 with AB302940 at 1/50 dilution (21.26 μg/ml), followed by ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor^®488) preadsorbed antibody at 1/1000 dilution (2 μg/mL) (Green). Confocal image showing cytoplasmic staining in MCF7 cell line. Recombinant Alexa Fluor^®594 Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab202272) was used to counterstain tubulin at 1/50 dilution (10 μg/mL) (Red). The Nuclear counterstain was DAPI (Blue). 

Secondary antibody only control: Secondary antibody is ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (2 μg/mL) . 

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PLCG1 KO HEK293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) (ab266530) cells labeling Phospholipase Cγ1 with AB302940 at 1/100 dilution (10.63 μg/ml), followed by ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (2 μg/mL) (Green). Confocal image showing cytoplasmic staining in parental HEK293T cell line and no staining in PLCG1 KO HEK293T cell line. Recombinant Alexa Fluor^®594 Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab202272) was used to counterstain tubulin at 1/50 dilution (10 μg/mL) (Red). The Nuclear counterstain was DAPI (Blue). 

Secondary antibody only control: Secondary antibody is ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor^®488) preadsorbed at 1/1000 dilution (2 μg/mL).