Anti-Midkine antibody [EP1143Y]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Midkine antibody [EP1143Y] (AB52637)

Flow cytometric overlay histogram of 4% paraformaldehyde-fixed, 90% methanol permeabilised SH-SY5Y (Human neuroblastoma epithelial cell line) positive cells (Left) and Ramos (Human Burkitt's lymphoma B lymphocyte cell line) negative cells (Right) labelling Midkine with ab52637 at 1/2000 dilution (red line). A Goat Anti-Rabbit IgG Alexa Fluor® 488 secondary antibody (ab150081) was used at 1/2000 dilution. Isotype control antibody (black line) was Rabbit monoclonal IgG (ab172730) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was cells without incubation with primary antibody and secondary antibody and was also used as a control.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Midkine antibody [EP1143Y] (AB52637)

ab52637 staining MDK in SH-SY5Y (positive) and HL-60 (negative) cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated for 2 hours at room temperature with ab52637 at 0.2 ug/ml (shown in green) and ab7291, Anti-alpha Tubulin antibody [DM1A] - Loading Control (shown in pseudocolour magenta). Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution and ab150119, Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed at 1/1000 dilution for 1 hour at room temperature. Nuclear DNA was labeled with DAPI (shown in blue). Image was acquired with a high-content analyser (Operetta CLS, Revvity) and a maximum intensity projection of confocal sections is shown.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Midkine antibody [EP1143Y] (AB52637)

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.

• IP

Unknown

Immunoprecipitation - Anti-Midkine antibody [EP1143Y] (AB52637)

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.

All lanes:

Immunoprecipitation - Anti-Midkine antibody [EP1143Y] (ab52637)

Predicted band size: 16 kDa

Observed band size: 16 kDa

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• WB

Lab

Western blot - Anti-Midkine antibody [EP1143Y] (AB52637)

Blocking and diluting : 5% NFDM/TBST

All lanes:

Western blot - Anti-Midkine antibody [EP1143Y] (ab52637) at 1/1000 dilution

All lanes:

SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 16 kDa

Observed band size: 16 kDa

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• WB

Collaborator

Western blot - 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.

All lanes:

Western blot - Anti-Midkine antibody [EP1143Y] (ab52637)

Predicted band size: 16 kDa

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• WB

Supplier Data

Western blot - Anti-Midkine antibody [EP1143Y] (AB52637)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Midkine can form SDS- and β-mercaptoethanol-resistant dimers and multimers (PMID : 9083079，PMID : 7721890.

All lanes:

Western blot - Anti-Midkine antibody [EP1143Y] (ab52637) at 1/100 dilution

All lanes:

SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 15-70 kDa

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Exposure time: 180s

• WB

Lab

Western blot - Anti-Midkine antibody [EP1143Y] (AB52637)

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.

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• WB

Lab

Western blot - Anti-Midkine antibody [EP1143Y] (AB52637)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Midkine antibody [EP1143Y] (ab52637) at 1/10000 dilution

All lanes:

Western blot - Recombinant Human Midkine protein (<a href='/ko/products/proteins-peptides/recombinant-human-midkine-protein-ab49848'>ab49848</a>) at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 16 kDa

Observed band size: 16 kDa

false

• WB

Unknown

Western blot - Anti-Midkine antibody [EP1143Y] (AB52637)

All lanes:

Western blot - Anti-Midkine antibody [EP1143Y] (ab52637) at 1/500 dilution

All lanes:

Recombinant protein at 0.001 µg

Secondary

All lanes:

Goat anti-rabbit HRP labeled at 1/2000 dilution

Predicted band size: 16 kDa

Observed band size: 16 kDa

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