All lanes : Anti-TGF beta 1 antibody [EPR21143] (ab215715) at 1/1000 dilution

Lane 1 : Wild-type HeLa whole cell lysate
Lane 2 : TGFB1 knockout HeLa whole cell lysate
Lane 3 : A549 whole cell lysate
Lane 4 : Mouse spleen tissue lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 44 kDa
Observed band size: 13,44 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab215715 observed at 13 and 44 kDa. Red - loading control, ab7291, observed at 50 kDa.

ab215715 was shown to specifically react with in wild-type HeLa cells as signal was lost in TGFB1 knockout cells. Wild-type and TGFB1 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% NF Milk. Ab215715 and ab7291 (Mouse anti-tubulin 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.

Immunohistochemical analysis of paraffin-embedded human thrombocytosis tissue labeling TGF beta 1 with ab215715 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic staining of megakaryocytes in human thrombocytosis (PMID: 25305163). Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

 

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling TGF beta 1 with ab215715 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining of megakaryocytes and platelets in rat spleen (PMID: 25305163). Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling TGF beta 1 with ab215715 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining of megakaryocytes and platelets in mouse spleen (PMID: 25305163). Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

All lanes : Anti-TGF beta 1 antibody [EPR21143] (ab215715) at 1/1000 dilution

Lane 1 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 20 µg
Lane 2 : L-929 (mouse connective tissue fibroblast cell line) whole cell lysate at 20 µg
Lane 3 : A549 (human lung carcinoma cell line) whole cell lysate at 20 µg
Lane 4 : HL-60 (human promyelocytic leukemia cell line) whole cell lysate at 20 µg
Lane 5 : RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 20 µg
Lane 6 : Mouse spleen lysate at 10 µg

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

Developed using the ECL technique.

Predicted band size: 44 kDa
Observed band size: 12,44 kDa why is the actual band size different from the predicted?

 

Exposure times: Lanes 1-4: 3 minutes; Lane 5: 41 seconds; Lane 6: 24 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

The molecular mass observed is consistent with the literature (PMID 2139036).

The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument.