BD, 556030, BD Pharmingen™ FITC Mouse Anti-Human PCNA Set

Reactivity: Human (QC Testing)
Application: Intracellular staining (flow cytometry) (Routinely Tested)
Regulatory Status: RUO
RRID: AB_396306
Description: Description The P roliferating C ell N uclear A ntigen (PCNA) was initially identified as a nuclear antigen in proliferating cells and was subsequently described as a subunit for DNA polymerase δ.  PCNA protein levels peak during the S-phase of the cell cycle, at which time it forms a complex with the p21 inhibitor. PCNA is almost undetectable in other phases of the cycle. Because of its unique expression, PCNA has been extensively used in studies associating the prognosis of tumor progression and neoplastic proliferation.  Human PCNA has been reported to be 262 amino acids with an apparent molecular weight of 36 kDa.
Preparation And Storage: Preparation And Storage The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with FITC under optimum conditions, and unreacted FITC was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
Recommended Assay Procedures: Recommended Assay Procedures 1. Harvest, count and pellet cells following standard procedures. Note: PCNA is expressed by proliferating cells. Using resting cells (eg, unstimulated PBMC) may give negative results. 2. While vortexing, add 5 ml cold 70% - 80% ethanol dropwise into the cell pellet (1-5 x 10^7 cells). Incubate at -20°C for at least 2 hours. These fixed cells can be stored at -20°C for up to 60 days prior to staining. 3. Wash twice with 30-40 ml staining buffer (PBS with 1% FBS, 0.09% NaN 3 ), centrifuge for 10 minutes at 200g. 4. Resuspend the cells to a concentration of 1 X 10^7/ml. 5. Transfer 100 µl (1 X 10^6 cells) cell suspension into each sample tube. 6. Add 20 µl of properly diluted anti-PCNA antibody according to the protocol into the tubes above. Mix gently. 7. Incubate the tubes at room temperature (RT) for 20-30 minutes in the dark. 8. Wash with 2 ml of staining buffer at 200g for 5 minutes. 9. Aspirate the supernatant. 10. If using directly conjugated anti-PCNA, proceed to step 13. 11. If using purified anti-PCNA, add 50 µl of diluted secondary antibody (eg, Cat. No. 555988), if using Biotin conjugated anti-PCNA, add 50 µl of SAV-PE  (Cat. No. 554061), to each sample tube and incubate at RT for 30 minutes in the dark. 12. Repeat steps 8 & 9. 13. Add 0.5 ml of staining buffer to each tube. If using FITC conjugated anti-PCNA or secondary antibody, add 10 µl of Propidium Iodide Staining Solution (Cat. No. 556463) to each tube; for PE conjugated anti-PCNA or secondary antibody, add 20 µl BD Via-Probe™ Cell Viability Solution (Cat. No. 555816) to each tube. 14. Proceed to flow cytometric analysis.