TIG - - February 1986

Electroelution of proteins from SDS gels

Cloning a eukaryotic gene often demands prior purification of the protein so that either antibodies can be raised to screen an expressien hq~ary or oligo- nudeotide probes can be de- signed from protein sequence data for hybridization screening. Furthermore, once a DNA se- quence is obtained it is unwise to rely on this alone for predicting the protein sequence; it should be checked directly. Rapid iso- lation of protein in sufficient quantity and purity can present problems for non-specialist lab- oratories. In most molecular biology laboratories SDS-PAGE is routinely used and has the potential for purifying proteins from complex mixtures. How- ever, a m,qor problem is the efficient recovery of undegraded protein from the geL Electro- elution provides an answer but present methods use complex expensive apparatus, are leng- thy and not suitable for multiple samples 1.

We have adapted a technique nommlly used for electroeluting DNA from agarose gels z to the recovery of proteins from SDS- PAGE gels. It merely requires an inexpensive flat-bed electro- phoresis tank of the type avail- able iq all molecular biology laboratories. To date, at least 40 proteins have been purified by this method, ranging in size from 3.5 to 100 kDa and including both membrane and giycopro- teins. The purified proteins have been used to raise monospecific polydonal ant~xlles and for amino acid composition and protein sequence analysis.

After conventional SDS- PAGE 3, stain the gel with 0.1% Coomassie blue R250 in fixative (10% acetic acid, 30% methanol) and then destain in fixative (10rain each step) (Ref. 4). Excise the visualized protein bands and seal them in small dialysis bags (V'mldng tubing, or Spectropor for proteins <10kDa) with just enough

technical tips elution buffer (0.1 M sodium phosphate, pH 7.4, 0.1% SDS) to cover the gel pieces. Place the bags on the platform of the gel tank, cover them with elution buffer and electroelute (100 mA, 20-200 V) as for DNA (Ref. 2). Either microfuge (10 min) or uitrafdter (Durapore membrane) the electroeluted protein solu- tion to remove acrylamide debris, dialyse extensively at 4°(: against water (5 × 5 I) and lyophilise. For convenience, electroelute overnight, but 2-8 h is often sufficient. The s t a ~ g d e ~ which should be brief for quantitative (~80%) recovery, is sensitive down to -0.1 pg/band. Residual stain is uuimpo~mt and helps monitor electroelution. We have also successfully electroeluted radio- labelled proteins (< 1 ng) de- tected by autoradiography. Where acid fixation is undesir- able, (to avoid destruction of sensitive residues, e.g. meth- ionine) sodium acetate visual- ization s can be substituted. Typical gel apparatus is suitable

for simultaneous electroelution of multiple samples. For mem- brane proteins, up to 1~ SDS can be t~sed for solubility during electvoelution and all but f,~J dialysis change.

Contributed by Chm'les McDonald*, Stephen Fawell, Darryl Pappin and Stephen Higghm, Departments of Biochemistry, Universities of Sheffield* (ShettieM S10 2TN) and Leeds (Leeds LS2 9JT), UFL

References I Hunkapill~, M. W., Lujon, E.,

Ostrander, F. and Hood, L. (1983) Methods En,,ymoi. 91, 227-236

2 McDonnell, M. W., Shnon, M. N. and Studier, F. W. (1977)]. Mol. Biol. 110, 119--146

3 Laemn~ U. I~ (1970)Nature227, 680-685

4 Cleveland, D. W., Fischer, S. G., Kirsdmer, M. W. and Laemmii, U. K. (1977)]. Biol. Chem. 252, 1102-1106

5 Higgins, R. L. and Dalunus, M. E. (1979) Anal. Biock~. 93, 257-260

Gene assembler* The Pharmacia Gene Assem- bler TM is a fully automated DNA synthesizer which is prepro- grammed for standard methods but can easily be reprogrammed by the operator. In addition, it has an integrated monitor with print -out which allows calculation of coupling efficiency during synthesis. If the coupling is incomplete the machine auto- matically shuts down, saving reagents and work.

The chemistry chosen for standard methods is the phos- phite method with [~-cyanoethyl- amidites providing high coupling efficiency (>98%) with short cycle times (9.7 min). The high coupling efficiency together with the high reservoir capadty gives a great potential for synthesizing long (>100 bases) oligonucleo- tides. The s~thesis is per- formed on a support of Mono- Beads TM prepacked in dispos- able color-coded cassettes which are loaded into the two column reactors.

Pharmacia, Molecular Biology Divi- sion, 5-75182, Uppsala, Sweden.

* Based on information provided by the manufacturer.

Isolation of total nucleic acids from tissues and cells

This procedure for the isolation thiocyanate and the RNA select- of biologically active poly(A) + ively precipitated from the mRNAs, poly(A)- RNAs and homogenate with LiBr. DNA DNA from cells and tissues is subsequently recovered from claimed to be simple and effid- the soluble phase by ethanol ent. Tissue is rapidly homogen- precipitation is further purified ized or cells lysed in guanidine by RNase A digestion, protein-

ase K treatment and phenol- chloroform extraction. RNAs are purif~ from the precipitate by guanidine-HC! extraction and separated into poly(A) + and poly(A)- RNAs by oligo(dT)- cellulose chromatography.

Krawetz, S. A., States, J. C. and Dixon, G. H, (1986)]. Bioc~m. Bi~kys. Methods 12, 29-36

Horizontal electroblotfing

In this wire electrode arrange- ment for a horizontal electro- blotting apparatus the electredes are mounted in electrode con- duits built on perforated pie]d- glass plates. Electroblotfing is carried out in a buffer tank and proteins are separated by so- dium dodecyl sulfate-polyacryl- amide gel electrophoresis and electrophoretically transferred onto nitrocellulose membranes. Gas bubbles developed by elec- trolysis at the lower electrode are trapped in electrode conduits and removed by circulating buffer. Accumulation of gas bubbles between the electrodes during operation is thus avoided.

Laufitzen, E. (1986)]. B/ockem. Bio~ys. Methods 12, 113-120

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