Anti-CSN7b antibody [EPR6464]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CSN7b antibody [EPR6464] (AB133548)

Immunohistochemical analysis of paraffin embedded Human kidney tissue labelling CSN7b with ab133548 antibody at a dilution of 1/250.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CSN7b antibody [EPR6464] (AB133548)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling CSN7b with unpurified ab133548 at 1/200 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti-rabbit IgG (Alexa Fluorr^® 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.

• WB

Lab

Western blot - Anti-CSN7b antibody [EPR6464] (AB133548)

Lanes 1-4 : Merged signal (red and green). Green - ab133548 observed at 32 kDa. Red - loading control ab8245 observed at 36 kDa.

ab133548 Anti-CSN7b antibody [EPR6464] was shown to specifically react with CSN7b in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266646 (knockout cell lysate ab257895) was used. Wild-type and CSN7b knockout samples were subjected to SDS-PAGE. ab133548 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and 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-CSN7b antibody [EPR6464] (ab133548) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

COPS7B knockout HEK293T cell lysate at 20 µg

Lane 2:

Western blot - Human COPS7B (CSN7b) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-cops7b-csn7b-knockout-hek-293t-cell-line-ab266646'>ab266646</a>)

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

HAP1 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 30 kDa

Observed band size: 32 kDa

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• WB

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Western blot - Anti-CSN7b antibody [EPR6464] (AB133548)

All lanes:

Western blot - Anti-CSN7b antibody [EPR6464] (ab133548) at 1/10000 dilution

Lane 1:

Jurkat cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 30 kDa

Observed band size: 30 kDa

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• WB

Lab

Western blot - Anti-CSN7b antibody [EPR6464] (AB133548)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : CSN7b knockout HAP1 cell lysate (20 μg)
Lane 3 : HeLa cell lysate (20 μg)
Lanes 1 - 3 : Merged signal (red and green). Green - ab133548 observed at 32 kDa. Red - loading control, ab18058, observed at 124 kDa.
ab133548 was shown to specifically react with CSN7b when CSN7b knockout samples were used. Wild-type and CSN7b knockout samples were subjected to SDS-PAGE. ab133548 and ab18058 (loading control to vinculin) were both diluted 1/10000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CSN7b antibody [EPR6464] (ab133548)

Predicted band size: 30 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-CSN7b antibody [EPR6464] (AB133548)

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.