The composition and preparation steps of the two lysates.

related topics .The components of cytolytic lysate include Tris-HCl,
EDTA
and NaCl.
the
lysate are mainly Urea, thiourea, CHAPS, etc. In addition to protein extraction lysate, the use of these two lysates in some experiments is not to be underestimated. This paper describes the formulation and formulation of these two lysates.tissue lysate formula: '7M Urea (60. 06) 4.2042 g 2M thiourea (76.12) 1.5224 g 100mM
DTT
0.1543 g 4% CHAPS 0.4 g 0.5mM EDTA 0.00146 g 40Mm Tris 0.0485 g 2% (v/v) NP-40 0.2 ml 1% (v/v) Triton X-100 0.. 1 ml5mM PMSF plus 0.00871 g 2% pharmalyte 0.2ml10ml in 400μl/tube. (Can be applied to 30-80 mg tissue cleavage) .Note: It has been packed into 400 μl/tube for applicationno additional !!!cell lysate formula: 6M Urea (60.06) 1.8018 g 2M thiourea (76.12) 0. 7613 g 65mM DTT 0.050 g 4% CHAPS 0.2 g 40Mm Trisbase 0.02425 g5ml in 200 sl/tube (available for 50-100ml
culture
bottles) Solution 1 , solution preparation 2× sample buffer: 130mM Tris-HCl (pH8.0), 20% (v/v) glycerin, 4.6% (w/v)
SDS
, 0.02% bromophenol blue, 2% DTT PBS ( pH7.4) : 10mM Na2HPO4, 1.8mM KH2PO4, 50mM NaCl, 2.7mM KCl RIPA Buffer: 50mM Tris-HCl (pH7.4) , 150mM NaCl, 1mM EDTA, 1% Triton×100, 1% sodium deoxycholicate, 0.1% SDS, 1mM PMSF, 1 sg/ml bovine pancreas
protease

inhibitors ( aprotinin ) , 1 sg/ml bright protease peptides ( leupeptin )lysate buffer: 10mM Tris-HCl (pH7.4), 0.15M NaCl , 5mM EDTA (pH8.0), 1% Triton×10
ε 0;
preparation of cell lysate: .Collect the cells digested by trypsin and centrifuge them and wash them with PBS.10min (20 sl RIPA buffer per 500,000 cells) in an ice bath with 100 sl RIPA buffer.10,000 rpm, centrifuged at 4 degrees C 10min, take the clean transfer to the new tube.the concentration of proteins in the protein was detected
the
blue method.the solution concentration to 5 mg/ml with a lysed buffer, and store it at 100 μl, -80C per vial. sample buffer is mixed with a 1:1 × ratio, boiling 5min and placing 5min at room temperature. electrophoresis test after short-term centrifuge. preparation tissue lysate: the solution or tissue is placed on ice throughout the preparation process. cut the tissue, saying it was cleaned in PBS with 1.5g. the tissue in the RIPA buffer, transfer to the homogenizer, add 5 ml of RIPA buffer and grind for 20min. the grinding fluid into a 1.5 ml centrifuge tube, 14000 rpm, 4 degrees C centrifugation 10min. the liquid as a complete tissue lysate. protein concentration was determined using the Coomas bright blue method. the solution concentration to 5 mg/ml with a lysed buffer, and store it at 100 μl, -80C per vial. sample buffer is mixed with a 1:1 × ratio, boiling 5min and placing 5min at room temperature. electrophoresis test after short-term centrifuge. it is important to note that in order to reduce and eliminate
nucleic acid
degradation, the sample is thoroughly homogenized before the steps need to be paid more attention. There is also a sample electrophoresis test after a short period of centrifuge. Make the sample from the original living environment time as short as possible
.