|Datasheet||Specific References||Reviews||Related Products||Protocols|
|Baculovirus-Insect Cells transfected lysate in which Human PCNA has been over-expressed. The whole cell lysate is provided in 1X Sample Buffer (1X modified RIPA buffer+1X SDS sample buffer).|
|Cell lysate was prepared by homogenization in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined with Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 minutes in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.|
|Modified RIPA Lysis Buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 1% Sodium deoxycholate, 1mM PMSF|
|12.5% SDS-PAGE Stained with Coomassie Blue|
|Samples are stable for up to twelve months from date of receipt at -80℃|
|1. Centrifuge the tube for a few seconds and ensure the pellet at the bottom of the tube. 2. Re-dissolve the pellet using 200μL pure water and boiled for 2-5 min. 3. Store it at -80℃. Recommend to aliquot the cell lysate into smaller quantities for optimal storage. Avoid repeated freeze-thaw cycles. Notes：The lysate is ready to load on SDS-PAGE for Western blot application. If dissociating conditions are required, add reducing agent prior to heating.|
|In modified RIPA Lysis Buffer|
|Store at -80℃. Aliquot to avoid repeated freezing and thawing|
|WB: Use at an assay dependent dilution.|
Not yet tested in other applications.
Optimal dilutions/concentrations should be determined by the end user.
Proliferating Cell Nuclear Antigen (PCNA) is a protein only expresse in nomal proliferate cells and cancer cells. It is central to both DNA replication and repair. One of the well-established functions for PCNA is its role as the processivity factor for DNA polymerase delta and epsilon. PCNA tethers the polymerase catalytic unit to the DNA template for rapid and processive DNA synthesis. Two forms of PCNA exist in cells: (i) a detergent-insoluble trimeric form stably associated with the replicating forks during S phase and (ii) a soluble form in quiescent cells in G1 and G2 phases. PCNA forms a toroidal trimer in S phase with replication factor-C (RF-C) and DNA in an ATP-dependent manner and enables the loading of DNA polymerase delta and epsilon onto the complex. The close association of PCNA with kinase complexes involved in cell cycle machinery indicates that PCNA has a regulatory role in cell cycle progression. PCNA also participates in the processing of branched intermediates that arise during the lagging strand DNA synthesis.