RIPA buffer

Nonspecific interacting proteins are removed by washing the beads thrice with modifiedradioimmune precipitation assay buffer (RIPA) and once with phosphate-buffered saline. 

In Protocol IB, a soluble whole cell extract is produced by lysis in RIPA, a mixed micelle detergent-containing buffer. RIPA buffer results in extraction of proteins without complete denaturation and cellular antigens are maintained in conformations that can be detected by immrmopredpitation (Protocol 14B). How-... 

In Protocol 14C, immunoprecipitation is performed under disruptive conditions in a mixed micelle detergent buffer (RIPA). Most physiological interactions are dissociated imder these stringent conditions, and usually only the antigen protein and veiy tightly-associated proteins are recovered by immunoprecipitation. This method is useful to quantitate levels of pS]methionine labelled protein antigen, and to study synthesis and degradation rates by pulse chase analysis. 

After 2 h incubation of the prepared antibody beads with UV-crosslinked extract in a cold room, the beads are washed 4 x with 100 /A RIPA buffer (50 mMTris-HCl pH 7.5, 150 rnMNaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) and lx with genomic DNA lysis buffer (50 mM Tris, pH 7.4, 10 mM EDTA, 500 mM NaCl, 2.5 mM DTT, 0.5 mM spermidine, 1% Triton X-100). Approximately 300 /(I of PK solution (1 mg/ml proteinase K in genomic DNA lysis buffer and 0.2 U//A RNase inhibitor) is added to the total lysate previously kept on ice and the beads are then incubated at 37° for 30 min. Gently flick the tubes to resuspend the beads every 10 min during the incubation. After removal of the proteinase K solution, 300 /A of RNA extraction solution (4 M guanidine thiocyanate, 0.5% sarkosyl, and 25 mM sodium citrate, pH7) is added to the beads, incubated for 10 min and the supernatant is mixed with 30 fig yeast tRNA (as a carrier) and 30 fil of 3 M sodium acetate. The RNA solution is phenol-chloroform extracted, ethanol-precipitated, and the pellet washed once with 70% ethanol. The dry pellet is used for 1st strand cDNA synthesis, followed by PCR analysis. The removal of proteins... 

RIPA lysis buffer (without vanadate and sodium fluoride inhibitor as they inhibit phosphatase activity)... 

Lyse the cells in RIPA buffer without vanadate or NaF (lysis buffer). 

Wash the precipitates twice with the RIPA buffer, followed by one wash with the IX phosphatase assay buffer. 

RIPA lysis buffer, pH 8.0 Undiluted Diluted 1 40 Not tested... 

Cell lysis is required when using the ELISA, TR-FRET, or AlphaScreen readouts to detect the relative quantity of a specific intracellular phosphorylated protein sequence. A typical 2X to 4X RIPA buffer (commonly used for immunoprecipitation) containing a non-ionic detergent such as NP-40 or Tween20 is used. The lysis solution should contain protease and phosphatase inhibitors so that all further metabolism of the analyte is stopped. Often, an MTP shaking step is required to efficiently lyse the cells. 

Discard the supernatant and immediately add 300 pL of RIPA lysis buffer complemented with protease and PhosphoSTOP phosphatase inhibitors. 

To perform an immunoprecipitation (IP) experiment, a minimum of20x 10 cells are needed. Lyse the cells in 1 mL RIPA buffer and collect supernatants in fresh 1.5 mL microtubes. 

Set up immunoprecipitation by combining chromatin with 2X RIPA-POL buffer, TE and Dynabeads bound to primary antibody so as to make final concentration IX RIPA-POL in a total volume of 500 jL. 

Aspirate nnbound chromatin using a Pasteur pipette on a vacuum. Wash bead complexes with 1 mL of freshly prepared IX RIPA-POL buffer. Add buffer to each tube and invert tubes twice to resuspend. 

Cells were treated in T75 flasks and lysed in radio-immuno-precipitation assay (RIPA) buffer (50 mM Tiis-HCl, pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 2 mM EDTA, protease inhibitor complete minitab and 10 mM sodium orthovanadate). 500 pg of homogenate was incubated with 5 pg anti-Ret antibodies in TBS-T buffer (20 mM Tri HCl, pH 7.6, 137 mM NaCl, and 0.1% Tween 20) overnight at 4°C, followed by 2 h incubation with Protein G agarose. Samples were separated on an SDS-PAGE gel for Western blot analysis with anti-pTyr and anti-Ret antibodies. 

Wash the beads four times with PBS containing 1% NP-40 (instead of PBS, RIPA buffer may be used for more stringent washing). Wash the beads two times with a buffer containing 10 mM Tris-HQ, pH 8.0, 0.25 M LiQ, 0.5% NP-40, 0.5% Na deoxycholate, 1 mM EDTA. Wash the beads two times with TE. Resuspend the beads in 120 fil of TE. Remove 10 /il for Western analysis. Remove 10 fil and count [ Hjthymidine. This will give an idea of how efficient the immunoprecipitation was, and should show a large difference between preimmune and anti-SATBl serum. 

Add 40 pi of RIPA lysis buffer containing protease and phosphatase inhibitors. Lysis is allowed to proceed for 45 min on ice. 

The radiolabeled medium is removed, and the embryonic tissue is washed once in cold medium before being dissociated in either 2X dissociation buffer or RIPA/ NP40 lysis buffer. 

Embryos or cell cultures are collected and washed in PBS prior to homogenization using a dounce homogenizer, or other commercial disruption device, with ice-cold RIPA or NP40 lysis buffer. 

Spin down the beads, and add 20 mM ethanolamine, pH 8.2, to stop the reaction. After 5 min, wash three times with 0.1 M borate buffer, and finally resuspend the coupled beads in RIPA or NP40 lysis buffer (Note 7). 

Wash beads up to five times with lOx bead volume of cell or tissue lysis buffer by adding buffer, centrifuging at 1500 X g at 4°C for 1 min, and removing supernatant. (Note To retain weakly interacting proteins, use a low stringency wash buffer, such as PBS or 0.5% NP-40, add lOx bead volume, and briefly invert the tube 10 times [17]. Radioimmunoprecipitation assay [RIPA] buffer may also be used to retain more strongly interacting proteins.)... 

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