All lanes : Anti-CSN7b antibody [EPR6464] (ab133548) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : COPS7B knockout HEK293T cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : HAP1 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 30 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?

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.

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.

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 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.

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.

All lanes : Anti-CSN7b antibody [EPR6464] (ab133548) at 1/10000 dilution

Lane 1 : Jurkat cell lysate
Lane 2 : HeLa cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 30 kDa
Observed band size: 30 kDa

Equilibrium disassociation constant (K_D)
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