All lanes : Anti-CARM1 antibody (ab128851) at 1 µg/ml

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : CARM1 knockout HEK-293T cell lysate
Lane 3 : HT-29 cell lysate
Lane 4 : Caco-2 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 66 kDa
Observed band size: 65 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab128851 observed at 65 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab128851 was shown to react with CARM1 in wild-type HEK-293T cells in western blot with loss of signal observed in CARM1 knockout cell line ab266557 (CARM1 knockout cell lysate ab257871). Wild-type and CARM1 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab128851 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at 1 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

ICC/IF image of ab128851 stained DU145 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab128851 at 1µg/ml overnight at +4°C. The secondary antibody (green) was DyLight^® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

All lanes : Anti-CARM1 antibody (ab128851) at 1/500 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : CARM1 knockout HEK293T cell lysate
Lane 3 : HeLa 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: 66 kDa
Observed band size: 66 kDa

Lanes 1-3: Merged signal (red and green). Green - ab128851 observed at 66 kDa. Red - loading control ab8245 observed at 36 kDa. 

 ab128851 Anti-CARM1 antibody was shown to specifically react with CARM1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266557 (knockout cell lysate ab257871) was used.  Wild-type and CARM1 knockout samples were subjected to SDS-PAGE.  ab128851 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 500 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-CARM1 antibody (ab128851) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate

Lane 3 : MDA-MB-231 (Human breast adenocarcinoma cell line) Whole Cell Lysate

Lane 4 : HeLa Histone Preparation Nuclear Lysate

Lane 5 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate

Lane 6 : PC3 (Human prostate carcinoma cell line) Whole Cell Lysate Whole Cell Lysate
Lane 7 : DU 145 (Human prostate carcinoma cell line) Whole Cell Lysate


Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 66 kDa


Exposure time: 30 seconds

This antibody was raised against an immunogen that is predicted to recognize isoforms 1 and 3 of CARM1. The predicted molecular weights of isoforms 1 and 3 are 63 kDa and 66 kDa respectively.