Anti-CD168 antibody [EPR4054]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD168 antibody [EPR4054] (AB124729)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human gastric carcinoma tissue sections labeling CD168 with purified ab124729 at 1 : 250 dilution (7.7 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer pH9.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1 : 0 dilution. PBS instead of the primary antibody was used as the negative control.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD168 antibody [EPR4054] (AB124729)

Immunohistochemical analysis of paraffin-embedded human testis tissue using unpurified ab124729 at 1/50 - 1/100 dilution

• IP

Lab

Immunoprecipitation - Anti-CD168 antibody [EPR4054] (AB124729)

ab124729 (purified) at 1 : 100 dilution (2ug) immunoprecipitating CD168 in MCF-7 (Human breast adenocarcinoma epithelial cell) whole cell lysate.

Lane 1 (input) : MCF-7 (Human breast adenocarcinoma epithelial cell) whole cell lysate 10ug

Lane 2 (+) : ab124729 & MCF-7 (Human breast adenocarcinoma epithelial cell) whole cell lysate

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab124729 in MCF-7 (Human breast adenocarcinoma epithelial cell) whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1 : 1000 dilution.

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-CD168 antibody [EPR4054] (ab124729)

Predicted band size: 84 kDa

Observed band size: 85 kDa

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• WB

Lab

Western blot - Anti-CD168 antibody [EPR4054] (AB124729)

5% NFDM/TBST

All lanes:

Western blot - Anti-CD168 antibody [EPR4054] (ab124729) at 1/1000 dilution

Lane 1:

MDA-MB-435 (Human mammary gland ductal carcinoma melanocyte) whole cell lysates at 20 µg

Lane 2:

RAW264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates at 15 µg

Lane 3:

PC-12 (Rat adrenal gland pheochromocytoma ) whole cell lysates at 15 µg

Secondary

All lanes:

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 1/20000 dilution

Predicted band size: 125 kDa,84 kDa

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• WB

Lab

Western blot - Anti-CD168 antibody [EPR4054] (AB124729)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : Empty
Lane 3 : CD168 knockout HAP1 whole cell lysate (20 μg)

Lanes 1 - 3 : Merged signal (red and green). Green - ab124729 observed at 90 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab124729 was shown to specifically react with CD168 when CD168 knockout samples were used. Wild-type and CD168 knockout samples were subjected to SDS-PAGE. ab124729 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at a 1/1000 dilution and 1/10000 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CD168 antibody [EPR4054] (ab124729)

Predicted band size: 84 kDa

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• WB

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Western blot - Anti-CD168 antibody [EPR4054] (AB124729)

All lanes:

Western blot - Anti-CD168 antibody [EPR4054] (ab124729)

Lane 1:

MDA-MB-435 cell lysate at 10 µg

Lane 2:

LnCaP cell lysate at 10 µg

Lane 3:

T47-D cell lysate at 10 µg

Predicted band size: 84 kDa

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• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-CD168 antibody [EPR4054] (AB124729)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.