All lanes : Anti-Proteasome 20S LMP7 antibody (ab3329) at 2 µg/ml

Lane 1 : HeLa whole cell extracts
Lane 2 : HeLa treated with IFN gamma (100ng/ml IFN gamma for 72h) whole cell extracts
Lane 3 : U-937 whole cell extracts
Lane 4 : Raji whole cell extracts
Lane 5 : Ramos whole cell extracts

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG (H+L) HRP conjugate at 1/2500 dilution

Observed band size: 20 kDa why is the actual band size different from the predicted?

Detected by chemiluminescence.

Immunocytochemistry/ Immunofluorescence analysis of HeLa cells labeling Proteasome 20S LMP7 with ab3329 at 5µg/ml. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 in TBS for 10 minutes, and blocked with 3% Blocker BSA in PBS for 15 minutes at room temperature. Cells were stained with or without Anti-Proteasome 20S LMP7 antibody (ab3329), at a concentration of 5µg/ml for 1 hour at room temperature, and then incubated with a Alexa Fluor^® 488 goat anti-rabbit IgG secondary antibody at a dilution of 1/1000 for 1 hour s at room temperature (both panels, green). Nuclei (both panels, blue) were stained with Hoechst 33342 dye.

Ab3329 staining Human normal skin. Staining is localized to cytoplasmic and nuclear compartments.
Left panel: with primary antibody at 1 ug/ml. Right panel: isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffer citrate pH 6.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS), then incubated with primary antibody for 20 minutes, and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required

Western blot of proteasome 20S LMP7 from HeLa cell extract using ab3329.

ICC/IF image of ab3329 stained MCF7 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab3329, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.