Rapid detection of octamer binding proteins with'mini-extracts', prepared from a small number of cells.

E Schreiber, P Matthias, MM Müller… - Nucleic acids …, 1989 - ncbi.nlm.nih.gov
E Schreiber, P Matthias, MM Müller, W Schaffner
Nucleic acids research, 1989ncbi.nlm.nih.gov
Octaner binding proteins (oct-i, oct-2A/B) are transcription factors which regulate
theexpression of immunoglobulin heavy chain genes and various housekeeping genes
(reviewed in 1). Here we present an extremely simple protocol, modifled from that of Lee et
al.(2), which allows one to make nuclear extracts from as few as 5 x 105 cells or from
lymphocytes isolated from 2 ml of peripheral blood (3). At thesame time RNA can beisolated
from the cytoplasmic fraction and further analysed, eg byNorthernblotting. DetectionoftheDNAbindingproteinsisperfor …
Octaner binding proteins (oct-i, oct-2A/B) are transcription factors which regulate theexpression of immunoglobulin heavy chain genes and various housekeeping genes (reviewed in 1). Here we present an extremely simple protocol, modifled from that of Lee et al.(2), which allows one to make nuclear extracts from as few as 5 x 105 cells or from lymphocytes isolated from 2 ml of peripheral blood (3). At thesame time RNA can beisolated from the cytoplasmic fraction and further analysed, eg byNorthernblotting. DetectionoftheDNAbindingproteinsisperformedbybandshift assay. Many different cell lines can be quickly screened for thepresence ofoctamer-binding and other transcription factors. Moreover," factor-induction" experiments can be conveniently performed in small scale cultures. In addition, this technique will allow clinical investigations using bioptic material or blood mononuclear cells, eg lymphocytes purified from 2 ml of peripheral blood (3). Typically, 0.5-1 x 106cells from tissue culture, from homogenized mouse spleen (about 0.5 g) or peripheral blood lymphocytes are collected, washed with 10 ml TBS (Tris buffered saline) and pelleted by centrifugation at 1500x gfor5 min. The pellet isresuspended in 1 ml TBS, transferred into an Eppendorf tube and pelleted again by spinning for 15 sec in a microfuge. TBS is removed and the cell pellet is resuspended in 400 1 cold buffer A (10 mM HEPES pH 7.9; 10 mM KCI; 0.1 mM EDTA; 0.1 mM EGTA; 1 mM DTT; 0.5 mM PMSF) by gentle pipetting in a yellow tip. The cells are allowed to swell on ice for 15 min, afterwhich 25 R1of a 10% solution of Nonidet NP-40 (Fluka) is added and the tube is vigorously vortexed for 10 sec. The homogenate is centifuged for 30 sec in amicrofuge. Thesupematantcontaining cytoplasm andRNAistransferredtoafreshtubecontaining 400 1l buffer B (10 mM Tris pH 7.5; 7 M urea; 1% SDS; 0.3 M NaAc; 20 mM EDTA) and 600 g1 phenol/chloroform (1: 1), mixedimmediately and stored at-20 C until it is convenient to further purify theRNAaccordingto (4). The nuclear pellet is resuspended in 50g1ice-cold buffer C (20 mM BEPES pH 7.9; 0.4 M NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM DTIT; 1 mM PMSF) and the tube is vigorously rocked at 4 IC for 15 min on a shaking platform. The nuclear extract is centrifuged for 5 min in a microfuge at 4 C and the supernatant (ca. 55 1) is frozen in aliquots at-70'C. Usually, 1-2 p1 of this extract (ca 2-4 jg protein) is used for a bandshift assay in the presence of 3 jgpoly dI dC as described in (5).
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