All lanes : Anti-Rab4 antibody [EPR3042] - Early Endosome Marker (ab108974) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : RAB4A knockout HeLa cell lysate

Lysates/proteins at 40 µg per lane.

Performed under reducing conditions.

Predicted band size: 24 kDa
Observed band size: 24 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab108974 observed at 24 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab108974 was shown to react with Rab4 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264781 (knockout cell lysate ab257624) was used. Wild-type HeLa and RAB4A knockout HeLa cell lysates were subjected to SDS-PAGE. ab108974 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 Dilution and a 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.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Rab4 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Human fetal brain cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab108974 observed at 24 kDa. Red - loading control, ab8245, observed at 37 kDa.

Unpurified ab108974 was shown to recognize Rab4 when Rab4 knockout samples were used, along with additional cross-reactive bands. Wild-type and Rab4 knockout samples were subjected to SDS-PAGE. Unpurified ab108974 and ab8245 (loading control to GAPDH) were both diluted 1/2000 and incubated overnight at 4°C. 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 1/10 000 dilution for 1 h at room temperature before imaging.

All lanes : Anti-Rab4 antibody [EPR3042] - Early Endosome Marker (ab108974) at 1/2000 dilution (purified)

Lane 1 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates
Lane 3 : Neuro-2a (Mouse neuroblastoma neuroblast) whole cell lysates
Lane 4 : Mouse brain lysates
Lane 5 : Rat brain 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: 24 kDa
Observed band size: 25 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer: 5% NFDM/TBST

Immunocytochemistry/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling Rab4 with purified ab108974 at 1:100 dilution (8.8μg/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). ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunofluorescent staining of Rab4 in HeLa cells, using unpurified ab108974 at a 1/100 dilution.

All lanes : Anti-Rab4 antibody [EPR3042] - Early Endosome Marker (ab108974) at 1/2000 dilution (unpurified)

Lane 1 : MCF7 cell lysates
Lane 2 : PC12 cell lysates
Lane 3 : Fetal brain cell lysates

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 24 kDa
Observed band size: 25 kDa why is the actual band size different from the predicted?