All lanes : Anti-PSMB5/MB1 antibody (ab3330) at 1/1000 dilution

Lane 1 : NCI-H1299 (Human lung carcinoma cell line) whole cell lysate
Lane 2 : HeLa (Human epithelial adenocarcinoma cell line) whole cell lysate
Lane 3 : Mouse liver cell lysate

Lysates/proteins at 25 µg per lane.

Observed band size: 23 kDa why is the actual band size different from the predicted?

Immunocytochemistry/Immunofluorescence analysis of PSMB5/MB1 (green) showing staining in the cytoplasm and nucleus of A431 (Human epidermoid carcinoma cell line) cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were incubated with ab3330 in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Immunocytochemistry/Immunofluorescence analysis of PSMB5/MB1 (green) showing staining in the cytoplasm and nucleus of BAEC (Bovine aortic endothelial cell line) cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were incubated with ab3330 in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Immunocytochemistry/Immunofluorescence analysis of PSMB5/MB1 (green) showing staining in the cytoplasm and nucleus of HeLa (Human epithelial adenocarcinoma cell line) cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were incubated with ab3330 in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

ab3330 (4µg/ml) staining PSMB5/MB1 in human colon using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining of nuclear/cytoplasmic compartment of the intestinal glands cells.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min 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, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

Immunofluorescence analysis of RGC5 cells, staining PSMB5/MB1 with ab3330 at 1/100 dilution.