Immunofluorescence analysis of 70% confluent log phase MDA-MB-231 (Human breast adenocarcinoma cell line) cells labeling Proteasome 20S C2/HC2 (green) with ab3325 at 2 µg/mL. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with ab3325 in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) secondary antibody, Alexa Fluor® 488 conjugate at 1/2000 dilution for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with DAPI. F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin. Panel d represents the merged image showing cytoplasmic localization. Panel e shows the control without primary antibody. The images were captured at 60X magnification.

All lanes : Anti-Proteasome 20S C2/HC2 antibody (ab3325) at 2 µg/ml

Lane 1 : MDA-MB-231 (Human breast adenocarcinoma cell line) whole cell lysate
Lane 2 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lane 3 : PC-3 (Human prostate adenocarcinoma cell line) whole cell lysate
Lane 4 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 5 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG (H+L) HRP cpnjugate at 0.4 µg/ml

Developed using the ECL technique.

Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel, XCell SureLock™ Electrophoresis System and Novex® Sharp Pre-Stained Protein Standard. Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate.

ab3325 (1µg/ml) staining Proteasome 20S C2/HC2 in human colon using an automated system (DAKO Autostainer Plus). Using this protocol there is strong cytoplasmic and nuclear staining.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH6.1 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.

Anti-Proteasome 20S C2/HC2 antibody (ab3325) at 3 µg/ml + CHO (Chinese hamster ovary cell line) whole cell lysate