All lanes : Anti-TGF beta 1 antibody [EPR18163] (ab179695) at 1/1000 dilution

Lane 1 : A549 cell lysate
Lane 2 : K562 cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : TGF beta 1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 44 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab179695 observed at 50 kDa. Red - loading control, ab8245 observed at 37 kDa.  

 ab179695 was shown to react with TGF beta 1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255439 (knockout cell lysate ab263799) was used. Wild-type and TGF beta 1 knockout samples were subjected to SDS-PAGE. ab179695 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-TGF beta 1 antibody [EPR18163] (ab179695) at 1/5000 dilution

Lane 1 : L-929 (Mouse connective tissue fibroblast cell line) whole cell lysate
Lane 2 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 3 : A549 (Human lung carcinoma cell line) whole cell lysate
Lane 4 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) 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: 44 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 3 minutes; Lane 2-4:30 seconds.

 

Lane 1 : Anti-TGF beta 1 antibody [EPR18163] (ab179695) at 1/5000 dilution
Lanes 2-5 : Anti-TGF beta 1 antibody [EPR18163] (ab179695) at 1/1000 dilution

Lane 1 : Mouse serum
Lane 2 : Mouse spleen lysate
Lane 3 : Rat kidney lysate
Lane 4 : Rat spleen lysate
Lane 5 : Rat serum

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: 44 kDa
Observed band size: 12.5,50 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1/2/3/5: 3minutes; Lane 4: 30 seconds.

The expression profile observed is consistent with what has been described in the literature (PMID:18390240; PMID:12717387; PMID:2139036).

All lanes : Anti-TGF beta 1 antibody [EPR18163] (ab179695) at 1/1000 dilution

Lane 1 : Human kidney lysate
Lane 2 : Human 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: 44 kDa
Observed band size: 12.5,50 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression profile observed is consistent with what has been described in the literature (PMID:18390240; PMID:12717387; PMID:2139036).

All lanes : Anti-TGF beta 1 antibody [EPR18163] (ab179695) at 1/1000 dilution

Lane 1 : Recombinant protein fragment human TGF beta 1
Lane 2 : Recombinant protein fragment human TGF beta 2
Lane 3 : Recombinant protein fragment human TGF beta 3

Lysates/proteins at 0.01 µg per lane.

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

Predicted band size: 44 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 1 second; Lane 2/3: 3 minutes.

Recombinant protein fragment human TGF beta 1 contains aa279-390 with a GST-tag. Recombinant protein fragment human TGF beta 2 contains aa303-414 with a GST-tag Recombinant protein fragment human TGF beta 3 contains aa301-412 with a GST-tag. These three fragment recombinant proteins were made in-house.