Recombinant Anti-Midkine antibody [EP1143Y] (ab52637)

ab52637 was shown to react with MDK in wild-type HAP1 cells in Western blot with loss of signal observed in a MDK knockout cell line. Wild-type HAP1 and MDK knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab52637 overnight at 4 °C at a 1/500 dilution. Blots were incubated with goat anti-rabbit HRP secondary antibodies at 0.2 μg/ml before imaging. These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Midkine with purified ab52637 at 1:100 dilution (10.5μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

ab52637 (purified) at 1:20 dilution (2ug) immunoprecipitating Midkine in SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate.

Lane 1 (input): SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate 10ug

Lane 2 (+): ab52637 & SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab52637 in SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:5000 dilution.

Blocking and diluting buffer: 5% NFDM/TBST.

Human liver carcinoma stained with unpurified ab52637 at 1/100 - 1/250 dilution

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human pancreatic carcinoma tissue sections labeling Midkine with purified ab52637 at 1:50 dilution (2.1 μg/ml). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using EDTA Buffer, pH9.0. Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1:500 dilution. PBS instead of the primary antibody was used as the negative control.

Blocking and diluting: 5% NFDM/TBST

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Midkine with purified ab52637 at 1/250 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control. 

HeLa cells stained with unpurified ab52637 at 1/250 - 1/500 dilution