Anti-CD40 antibody [EPR20540] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20540] - BSA and Azide free (AB271995)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab213205).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20540] - BSA and Azide free (AB271995)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab213205).

• WB

Lab

Western blot - Anti-CD40 antibody [EPR20540] - BSA and Azide free (AB271995)

This data was developed using ab213205, the same antibody clone in a different buffer formulation.

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.

All lanes:

Western blot - Anti-CD40 antibody [EPR20540] - BSA and Azide free (ab271995) at 1/2000 dilution

Lane 1:

Wild-type U-2 OS whole cell lysate at 20 µg

Lane 2:

CD40 knockout U-2 OS whole cell lysate at 20 µg

Lane 3:

Raji whole cell lysate at 20 µg

Predicted band size: 30 kDa

Observed band size: 42 kDa

false

• WB

Unknown

Western blot - Anti-CD40 antibody [EPR20540] - BSA and Azide free (AB271995)

This data was developed using ab213205, the same antibody clone in a different buffer formulation :

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).

All lanes:

Western blot - Anti-CD40 antibody [EPR20540] - BSA and Azide free (ab271995) at 1/2000 dilution

Lane 1:

Human tonsil lysate at 20 µg

Lane 2:

Human lymph node lysate at 20 µg

Lane 3:

Human lymphoma lysate at 20 µg

Lane 4:

Raji (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Lane 5:

Daudi (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Lane 6:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Secondary

Lanes 1 - 3:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/4000 dilution

Lanes 4 - 6:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 30 kDa

Observed band size: 42 kDa

false