All lanes : Anti-MAPK6/ERK3 antibody [EP1720Y] (ab53277) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : MAPK6 knockout HeLa cell lysate
Lane 3 : 2.4G2 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: 63 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab53277 observed at 90 kDa. Red - loading control ab8245 observed at 36 kDa.

ab53277 Anti-MAPK6/ERK3 antibody [EP1720Y] was shown to specifically react with MAPK6/ERK3 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264910 (knockout cell lysate ab257526) was used. Wild-type and MAPK6/ERK3 knockout samples were subjected to SDS-PAGE. ab53277 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney carcinoma tissue sections labeling MAPK6/ERK3 with Purified ab53277 at 1:100 dilution (13.52 µg/ml). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Purified)ImmunoHistoProbe one step HRP Polymer (ready to use) was used for detection. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

All lanes : Anti-MAPK6/ERK3 antibody [EP1720Y] (ab53277) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : MAPK6 knockout HAP1 whole cell lysate
Lane 3 : A431 whole cell lysate
Lane 4 : MCF7 whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 63 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab53277 observed at 90 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab53277 was shown to react with MAPK6 in HAP1 wild-type cells in Western blot. Loss of signal was observed when MAPK6 knockout sample was used. HAP1 wild-type and MAPK6 knockout whole cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween^®) before incubation with ab53277 and ab8245 (Mouse anti-GAPDH antibody [6C5]) 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) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-MAPK6/ERK3 antibody [EP1720Y] (ab53277) at 1/1000 dilution (Purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : Rat brain lysates
Lane 3 : C2C12 (Mouse myoblasts myoblast) whole cell lysates

Lysates/proteins at 15 µg per lane.

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

Predicted band size: 63 kDa
Observed band size: 105 kDa why is the actual band size different from the predicted?

The molecular weight observed is consistent with what has been described in PMID:30642948 and 30166347

Unpurified ab53277 (1:50) staining human MAPK6/ERK3 in human breast carcinoma tissue by immunohistochemistry using paraffin embedded tissue.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.