Anti-CD40 antibody [EPR20540]

5 product images

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  Western blot - Anti-CD40 antibody [EPR20540] (ab213205)

  Lanes 1 - 3: Merged signal (red and green). Green - ab213205 observed at 42 kDa. Red - loading control, Anti-alpha Tubulin antibody [DM1A] - 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 Anti-alpha Tubulin antibody [DM1A] - Loading Control 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (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: Western blot - Anti-CD40 antibody [EPR20540] (ab213205) 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 2: Western blot - Human CD40 knockout U-2 OS cell line (Human CD40 knockout U-2 OS cell line ab262486)

  Lane 3: Raji (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 30 kDa

  Observed band size: 42 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20540] (ab213205)

  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) (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) (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.

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  Western blot - Anti-CD40 antibody [EPR20540] (ab213205)

  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] (ab213205) 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) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/4000 dilution

  Lanes 4 - 6: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 30 kDa

  Observed band size: 42 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20540] (ab213205)

  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) (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) (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.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20540] (ab213205)

  Tissue Microarrays stained for "Anti-CD40 antibody [EPR20540]" using "ab213205"in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0). The sections were incubated with ab213205 at +4°C overnight followed by Goat Anti-Rabbit IgG H&L (HRP) Goat Anti-Rabbit IgG H&L (HRP) ab97051 at 1/500.