Anti-CD40 antibody

• WB

Lab

Western blot - Anti-CD40 antibody (AB113701)

Lanes 1 - 3 : Merged signal (red and green). Green - ab113701 observed at 45 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab113701 was shown to react with CD40 in wild-type U-2 OS cells in Western blot. Loss of signal was observed when CD40 knockout cell line ab262486 (knockout cell lysate ab263923) was used. Wild-type and CD40 knockout U-2 OS cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab113701 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at 1 μg/ml and a 1 in 20000 dilution respectively. Blots were incubated 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 (ab113701) at 1 µg/mL

Lane 1:

Wild-type U-2 OS cell lysate at 40 µg

Lane 2:

CD40 knockout U-2 OS cell lysate at 40 µg

Lane 2:

Western blot - Human CD40 knockout U-2 OS cell line (<a href='/en-us/products/cell-lines/human-cd40-knockout-u-2-os-cell-line-ab262486'>ab262486</a>)

Lane 3:

Raji (Human Burkitt's lymphoma cell line) whole cell lysate at 40 µg

Predicted band size: 30 kDa

Observed band size: 45 kDa

false

• WB

Unknown

Western blot - Anti-CD40 antibody (AB113701)

CD40 contains a number of potential glycosylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted. The predicted molecular weight of CD40 is 31 kDa (SwissProt), however we expect to observe a banding pattern around 43 kDa. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.

All lanes:

Western blot - Anti-CD40 antibody (ab113701) at 1 µg/mL

Lane 1:

Human spleen tissue lysate - total protein (<a href='/en-us/products/unavailable/human-spleen-tissue-lysate-total-protein-ab29699'>ab29699</a>) at 10 µg

Lane 2:

Human thymus tissue lysate - total protein (<a href='/en-us/products/unavailable/human-thymus-tissue-lysate-total-protein-ab30146'>ab30146</a>) at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 30 kDa

Observed band size: 105 kDa,41 kDa,43 kDa

true

Exposure time: 10min

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody (AB113701)

IHC image of CD40 staining in Human normal spleen formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab113701, 5μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.