Boster Immunoleader

other

RIPA lysate solution

AR0105

50 ml

25 USD

AR0105

RIPA lysate solution

50 ml

25 USD

Western Blotting Related Reagent

Store at 4˚C for one year

Extraction of cellular proteins requires efficient cell lysis and protein solubilization, while avoiding protein degradation and/or interference with protein immunoreactivity and biological activity. RIPA (Radio-Immunoprecipitation Assay) Solution enables rapid, efficient cell lysis and solubilization of proteins from both adherent and suspension cultured mammalian cells. It has long been a widely used lysis and wash solution for small-scale affinity pull-down applications, such as immunoprecipitation, since most antibodies and protein antigens are not adversely affected by the components of this solution. In addition, RIPA Solution minimizes non-specific protein-binding interactions to keep background low, while allowing most specific interactions to occur, enabling studies of relevant protein-protein interactions.RIPA Solution is supplied as a ready to use solution that requires no preparation. Protease and phosphatase requires no preparation. Protease and phosphatase inhibitors may be added to the lysis solution as needed.

1． Cell sample 1) Pipette proper volume of RIPA solution and mix to well-distributed. Few minutes advanced before use, add PMSF buffer to make its final concentration to 1mM. 2) Anchorage-dependent cell: wash sample with PBS, NS or serum-free medium to remove culture solution. Add proper RIPA solution, then stroke with pipette to mix thoroughly. Shake slightly for 5-10 min. Then centrifuge at 10000-14000g for 10 min after ice incubation for 1-3h, collect the supernatants and conduct the follow-up steps. Instruction for RIPA USAGE SIZE of well/surface area Kit volume 100mm 500-1000ul 60mm 250-500ul 6-well plate 200-400ul per well 24-well plate 100-200ul per well 96-well plate 50-100ul per well 3) Suspending cell：After centrifuge, wash sample with PBS, NS or serum-free medium. Add RIPA solution, then stroke with pipette until cells separate. Vortex for 5-10 min and incubate on ice for 1-3h to lyses completely without obvious cell precipitation. Centrifuge at 10000-14000g for 10 min, collect the supernatants and conduct the next step. 2. Tissue Sample: 1) Put the tissue sample into precooling NS quickly and wash it several times to remove blood. Weight sample and cut it into small slices, then put them into tissue homogenizer. 2) Pipette proper volume of RIPA solution and mix to well-distributed. Few minutes advanced before use, add PMSF buffer to make its final concentration to 1mM. 3) Add RIPA Solution to tissues in 10:1 (RIPA lysate solution: tissue net weight = 10:1, i.e. add 10ml of RIPA lysate solution to 1g tissues) and homogenate. (If lyses incompletely, add more RIPA lysate solution; if high concentration protein samples are required, reduce the volume of lysate solution.) 4）Homogenate with glass homogenizer and incubate on ice for 1-3h until samples were lysed completely. 5) Centrifuge at 10000-14000g for 3-5 min, collect the supernatant and conduct the next step. Notes: 1. All steps of protein extraction should be operated on ice or at 4˚C. Aliquot the sample into sub-packages at proper volume, then freeze-drying or store at -20˚C in liquid form. Avoid freeze thawing repeatedly. 2. PMSF buffer is not included. Few minutes advanced before use, add PMSF buffer to make its final concentration to1mM. 3. After lysing, the protein sample contains detergent with high concentration. If Bradford detective method cannot work, try to use BCA Protein Assay Kit (AR0146) to detect the concentration.

For protein extraction in WB

process about 10g tissue or centrifuge and precipitate about 20ml cells