All lanes : Anti-Integrin beta 1 antibody [EPR16896] (ab179472) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ITGB1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 88 kDa
Observed band size: 140-150 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab179472 observed at 140-150 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab179472 was shown to react with ITGB1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255354 (knockout cell lysate ab263847) was used. Wild-type HeLa and ITGB1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab179472 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.

All lanes : Anti-Integrin beta 1 antibody [EPR16896] (ab179472) at 1/1000 dilution

Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : ITGB1 knockout HCT116 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 88 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?

Lanes 1 - 2: Merged signal (red and green). Green - ab179472 observed at 130 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab179472 was shown to react with Integrin beta 1 in HCT 116 wild-type cells in western blot with loss of signal observed in ITGB1 knockout cell line ab273724 (ITGB1 knockout cell lysate ab275251). HCT 116 wild-type and ITGB1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab179472 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 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.

All lanes : Anti-Integrin beta 1 antibody [EPR16896] (ab179472) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : Integrin beta 1 knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 88 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab179472 observed at 125 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab179472 was shown to specifically react with Integrin beta 1 in wild-type HAP1 cells as signal was lost in Integrin beta 1 knockout cells. Wild-type and Integrin beta 1 knockout samples were subjected to SDS-PAGE. Ab179472 and ab9484 (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-Integrin beta 1 antibody [EPR16896] (ab179472) at 1/1000 dilution

Lane 1 : Human fetal heart lysate
Lane 2 : Human fetal kidney lysate
Lane 3 : Human fetal spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/100000 dilution

Predicted band size: 88 kDa
Observed band size: 125 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression profile/ molecular weight  observed is consistent with what has been described in the literature (PMID: 23444218 and 23444218).

All lanes : Anti-Integrin beta 1 antibody [EPR16896] (ab179472) at 1/1000 dilution

Lane 1 : A431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 2 : U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate
Lane 3 : HT1080 (Human fibrosarcoma cell line) whole cell lysate
Lane 4 : U937 (Human histiocytic lymphoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 88 kDa
Observed band size: 125 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lane 1, 2 and 3: 3 seconds; Lane 4: 3 minutes.

The expression profile/ molecular weight  observed is consistent with what has been described in the literature (PMID: 23444218 and 23444218).

All lanes : Anti-Integrin beta 1 antibody [EPR16896] (ab179472) at 1/1000 dilution

Lane 1 : Rat kidney lysate
Lane 2 : Rat spleen lysate
Lane 3 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 4 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 5 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 88 kDa
Observed band size: 125 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lane 1 and  2: 3 minutes; Lane 3,4 and 5: 1 minute.

The expression profile/ molecular weight  observed is consistent with what has been described in the literature (PMID: 23444218 and 23444218).