All lanes : Anti-CD40 antibody [EPR20540] (ab213205) at 1/2000 dilution

Lane 1 : Wild-type U-2 OS whole cell lysate
Lane 2 : CD40 knockout U-2 OS whole cell lysate
Lane 3 : Raji whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 30 kDa
Observed band size: 42 kDa why is the actual band size different from the predicted?

Lanes 1 - 3: Merged signal (red and green). Green - ab213205 observed at 42 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab213205 was shown to react with CD40 in U-2 OS wild-type cells in Western blot. Loss of signal was observed when CD40 knockout sample was used. U-2 OS wild-type and CD40 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween®) before incubation with ab213205 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 2000 dilution and a 1 in 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 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CD40 with ab213205 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. 

Membranous and cytoplasmic staining on germinal center of human tonsil is observed [PMID: 10360965] [PMID: 7507299] [PMID:24452203].

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-CD40 antibody [EPR20540] (ab213205) at 1/2000 dilution

Lane 1 : Human tonsil lysate
Lane 2 : Human lymph node lysate
Lane 3 : Human lymphoma lysate
Lane 4 : Raji (Human Burkitt's lymphoma cell line) whole cell lysate
Lane 5 : Daudi (Human Burkitt's lymphoma cell line) whole cell lysate
Lane 6 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1-3 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/4000 dilution
Lanes 4-6 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 30 kDa
Observed band size: 42 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1,2,3 and 4: 30 seconds; Lane 5,6 and 7: 5 seconds.

The molecular weight observed is consistent with the literature (PMID: 23404288);

Negative control: Jurkat (PMID:10498643; PMID:15708600).

Immunohistochemical analysis of paraffin-embedded human large B cell lymphoma tissue labeling CD40 with ab213205 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Membranous staining on human large B cell lymphoma is observed [PMID: 7507299].

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.