All lanes : Anti-CD130 (gp130) antibody [EPR21732] (ab217671) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : IL6ST knockout A549 cell lysate
Lane 3 : PC3 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 104 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 - ab217671 observed at 130 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab217671 was shown to react with CD130 (gp130) in Wild-Type A549 cells in western blot. Loss of signal was observed when knockout cell line ab266939 (knockout cell lysate ab257208) was used. Wild-Type A549 and IL6ST knockout A549 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. ab217671 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.

ELISA analysis of IL6ST recombinant protein at 1000 ng/mL with ab217671. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as the secondary antibody.

All lanes : Anti-CD130 (gp130) antibody [EPR21732] (ab217671) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : IL6ST knockout A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 104 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 - ab217671 observed at 130 kDa. Red - loading control ab7291 observed at 50 kDa.

 ab217671 Anti-CD130 (gp130) antibody [EPR21732] was shown to specifically react with CD130 (gp130) in wild-type A549 cells. Loss of signal was observed when knockout cell line ab266938 (knockout cell lysate ab257207) was used. Wild-type and CD130 (gp130) knockout samples were subjected to SDS-PAGE. ab217671 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 1000 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-CD130 (gp130) antibody [EPR21732] (ab217671) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : IL6ST knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 104 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 - ab217671 observed at 130 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab217671 Anti-CD130 (gp130) antibody [EPR21732] was shown to specifically react with CD130 (gp130) in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265767 (knockout cell lysate ab257205) was used. Wild-type and CD130 (gp130) knockout samples were subjected to SDS-PAGE. ab217671 and Anti-GAPDH antibody [6C5] - Loading Control 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-CD130 (gp130) antibody [EPR21732] (ab217671) at 1/1000 dilution

Lane 1 : Wild-type HAP1 (human chronic myelogenous leukemia cell line) whole cell lysate
Lane 2 : CD130 (gp130) knockout HAP1 whole cell lysate
Lane 3 : PC-3 (human prostate adenocarcinoma 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

Developed using the ECL technique.

Predicted band size: 104 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?


Exposure time: 114 seconds

Blocking and dilution buffer: 5% NFDM/TBST.

ab217671 was shown to specifically react with CD130 (gp130) in wild-type HAP1 cells as the signal was lost in CD130 (gp130) knockout cells. Wild-type and CD130 (gp130) knockout samples were subjected to SDS-PAGE. ab217671 and ab181602 (Human anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/200000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument using the ECL technique.

All lanes : Anti-CD130 (gp130) antibody [EPR21732] (ab217671) at 1/1000 dilution

Lane 1 : Untreated PC-3 (human prostate adenocarcinoma cell line) whole cell lysate
Lane 2 : PC-3 whole cell lysate treated with PNGase F

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Developed using the ECL technique.

Predicted band size: 104 kDa
Observed band size: 100,130 kDa why is the actual band size different from the predicted?


Exposure time: 92 seconds

Blocking and dilution buffer: 5% NFDM/TBST.

The molecular masses observed are consistent with the literature (PMID 19915009; PMID 19056487).

All lanes : Anti-CD130 (gp130) antibody [EPR21732] (ab217671) at 1/1000 dilution

Lane 1 : U266B1 (human multiple myeloma B lymphocyte cell line) whole cell lysate at 20 µg
Lane 2 : Human fetal lung tissue lysate at 10 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Developed using the ECL technique.

Predicted band size: 104 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times.

Lane 1: 15 seconds.

Lane 2: 81 seconds.

The molecular mass observed is consistent with the literature (PMID 19915009; PMID 19056487).