All lanes : Anti-TRIM21/SS-A antibody [EPR20290] (ab207728) at 1/1000 dilution

Lane 1 : Wild-type A549 (Human lung carcinoma cell line) whole cell lysate
Lane 2 : TRIM21 knockout A549 (Human lung carcinoma cell line) whole cell lysate
Lane 3 : HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 4 : MOLT-4 (Human lymphoblastic leukemia cell line) whole 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: 54 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab207728 observed at 50 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab207728 Anti-TRIM21/SS-A antibody [EPR20290] was shown to specifically react with TRIM21/SS-A in wild-type A549 cells. Loss of signal was observed when knockout cell line ab267024 (knockout cell lysate ab257766) was used. Wild-type and TRIM21/SS-A knockout samples were subjected to SDS-PAGE. ab207728 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C 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-TRIM21/SS-A antibody [EPR20290] (ab207728) at 1/500 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : TRIM21 knockout A549 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : MOLT-4 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: 54 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab207728 observed at 50 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab207728 Anti-TRIM21/SS-A antibody [EPR20290] was shown to specifically react with TRIM21/SS-A in wild-type A549 cells. Loss of signal was observed when knockout cell line ab267025 (knockout cell lysate ab257767) was used. Wild-type and TRIM21/SS-A knockout samples were subjected to SDS-PAGE. ab207728 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-TRIM21/SS-A antibody [EPR20290] (ab207728) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : TRIM21 knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : MOLT-4 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 54 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab207728 observed at 50 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab207728 was shown to specifically react with in wild-type HAP1 cells as signal was lost in TRIM21 knockout cells. Wild-type and TRIM21 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% NF Milk. Ab207728 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-TRIM21/SS-A antibody [EPR20290] (ab207728) at 1/1000 dilution

Lane 1 : Untreated HeLa (human epithelial cell line from cervix adenocarcinoma), whole cell lysate
Lane 2 : HeLa (human epithelial cell line from cervix adenocarcinoma) treated with 10 ng/ml human interferon-a (ab48750) for 16 hours

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 54 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The level of TRIM21 expression can be elevated by IFN alpha treatment (PMID: 18071879).

All lanes : Anti-TRIM21/SS-A antibody [EPR20290] (ab207728) at 1/1000 dilution

Lane 1 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen), whole cell lysate
Lane 2 : MOLT-4 (human lymphoblastic leukemia cell line), whole cell lysate
Lane 3 : Human fetal spleen lysate
Lane 4 : Human fetal kidney lysate
Lane 5 : Human thymus lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 54 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-TRIM21/SS-A antibody [EPR20290] (ab207728) at 1/1000 dilution

Lane 1 : Rat spleen lysate
Lane 2 : Rat thymus lysate
Lane 3 : Mouse thymus lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 54 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

 

Exposure times: Lane 1-2: 30 seconds; Lane 3: 3 minutes.

Blocking/Dilution buffer: 5% NFDM/TBST.