Protein electroelution

There are a number of ways to separate ampholytes from proteins. Electroelution ammonium sulfate precipitation and gel filtration, ion exchange, and... 

Chloroform extraction has been shown to be effective in removing SDS from proteins electroeluted from SDS polyacrylamide gels [28]. After elution, the aqueous fractions were mixed with methanol and extracted with chloroform. The SDS was removed by the chloroform and the extracted protein was washed extensively with methanol and then dried to remove any residual chloroform. 

Preparative Electroelution of Proteins from Polyacrylamide Gels... 

Fill the gel particles together with the surrounding buffer into the tube. The tube is inserted into the vertical electrophoresis apparatus (dialysis membrane down to anode), electrode buffer is poured, and electroelution is started with 10 mA per tube for 3-8 h, depending on tbe molar mass of the protein. 

Since SDS is practically not dialyzable, it must be removed by other techniques extraction, precipitation, electroelution, and washing out from immobilized proteins. 

The SDS-containing solution is supplemented with an excess of a nonionic detergent, e.g., Triton X-100. The electrode buffer is a buffer with low ionic strength, without SDS, and pH nearby the pi of the protein of interest. During electroelution SDS migrates to the anode. 

The recombinant protein is eluted from the gel by using electroeluter Model 422. 

Figure 5-17 Electron micrograph of a six-noded knot made by the Tn3 resolvase which is involved in movement of the Tn3 transposon (Chapter 27) from one location to another within the genome. Putative six-noded knot DNA was isolated by electroelution from an agarose gel. The knots, which are nicked in one strand, were denatured to allow the nicked strand to slide away and leave a ssDNA knot. This was coated with E. coli rec A protein (Fig. 27-24) to greatly thicken the strand and to permit the sign of each node (designated in the tracing) to be seen. From Wasserman et a/.184...

An alternative technique is to extract and concentrate the proteins from the gel by electroelution (see Methods in Molecular Biology, Volume 1, Chapter 19), but this leads to considerable loss of material and low amounts of purified protein. 

Polyamide gel electrophoresis is certainly the method most often used for protein separation. This most powerful electrophoresis method combines a separation based on the isoelectric point in the first dimension and a size separation in the second dimension. Separation of several thousands proteins in one analytical operation is possible. The proteins to identify then are often presented to the mass spectrometrist as a spot or a band on a gel. The methods described before remain applicable but several dedicated modifications have been proposed. They differ by digestion step of the protein [92-94], Either the proteins are digested directly in gel, or they are extracted first by electroelution or electroblotting. 

Dunn, M.J. (1996a) Electroelution of proteins from polyacrylamide gels, Methods Mol. Biol. 59, 357-362. 

Unless otherwise indicated, proteins were electroeluted from single SDS-PAGE bands. 

The bottom-up approach very much resembles classical protein identification strategies. The proteins in the proteome are first separated by 2D-GE (Ch. 17.3), or in some cases by SCX, size-exclusion (SEC), or affinity (AfC) chromatography. Specific proteins are excised from the gel, blotted, or electroeluted. The protein is digested, and the digest is analysed by LC-MS. The EC separation involves either RPLC with microcapillary or nano-LC columns (Ch. 17.5.2), or 2D-LC with typically SEC or SCX in the first dimension and RPLC in the second (Ch. 17.5.4). Alternatively, the sample may be introduced via either direct-infusion nano-ESl (Ch. 17.2), CE-MS (Ch. 17.5.6), or a microfluidic device coupled to MS (Ch. 17.5.5). 

After the run it is advisable to stain the proteins with a careful method for example, with sodium acetate (see Section 1.4.2). You then cut out the interesting bands with a razor blade. Different devices for electroelution of proteins from gel pieces are available in retail. If you often use preparative electrophoreses, you should try the prep ceU from Bio-Rad. This device allows you to preparatively electrophorese and elude proteins in one step. 

Fig. 3 shows proteolytic fingerprints of the D-1 protein labeled by [ C]azido-monuron and the 38-41 kDa protein primarily tagged by [ H]2-acetoxymethyl-1,4-naphthoquinone. The proteins were electroeluted from PAGE gels and digested with Staphylococcus aureus V-8 protease. 

Pigment-protein complexes (CP) were isolated from 14h illuminated cells of mutant C-6D by polyacrylamide gel electrpjtoresis as described in Materials and Methods. Pigment composition was determined by HPLC after electroelution and extraction of complexes. Data are expressed as percent of total carotenoid content (neopneoxanthin, tri=trihydroxy-a-carotene, vio=violaxanthin, ant= antheraxanthin, lut=lutein, zea=zeaxanthin, a-c=30t-caro-tene, p-c=p-carotene). Chloroj yll a/b-ratio (a/b) and the ratio of xantho-phylls/carotenes (x/c) are also plotted. 

Figure 1. Purification of pea carbonic anhydrase. Tb figure shows a Coomassie Blue stained, 7-15% lineaf gradient, SDS polyacrlyamide gel (PAG) loaded with total soluble protein (lane G), 30-60% (NH4)2S04 precipitate (lane F), eluate from the affinity columt chromatography (lane E) and after excision and electroelution from an SDS-PAG of the affinity column eluate (lane D). Lane C contains the polypeptides found in fractions exhibiting CA activity after Sephadex G-200 chromatography of the affinity column eluate and lane B contains polypeptides electroeluted from a section of a native PAG stained for CA activity. A Western blot of the total soluble protein (lane G) probed with the CA polyclonal antisera is shown in lane I. Molecular weight markers (Bio-Rad Inc.) are shown in lanes A and H...

Electroblotting is the most commonly used method of transferring proteins from a gel to a membrane. The principal advantages are the speed and the completeness of transfer compared with diffusion or vacuum blotting. Electroelution can be achieved either by (1) complete immersion of a gel-membrane sandwich in a buffer (wet transfer) or (2) placing the gel-membrane sandwich between absorbent paper soaked in transfer buffer (semi-dry transfer). 

There are many situations in which one would like to recover proteins from acrylamide gels for further analysis. For example, polypeptides extracted from gels can be readily used to immunize rabbits or mice to prepare antibodies (see articles by Christian Huet and by Ariana Celis, Kurt Dejgaard, and Julio E. Celis). Here we present a simple protocol for electroeluting proteins from fixed, unstained and Coomassie brilliant blue-stained dry two-dimensional (2D) gels (lEF or NEPHGE). 

Sometimes, electroeluted proteins give rise to artifactual variants. 

We have developed a rapid method for the preparation of the 3.6 kD peptide from bean seed using selective solvent extractions and preparative slab gel electrophoresis followed by electroelution. This method produces a product free of any detectable protein or peptide contamination. Using peptide produced in this way... 

A. technique not as commonly used as chemical methods it can none the less provide good yields of eluted antigen in a small volume without subjecting it to extremes of pH or denaturants (40, 41). It requires the use of additional apparatus, either home-made or commercial (e.g. Bio-Rad Model 422 electro-eluter). The washed matrix canying the adsorbed antigen has to be transferred from the column to an electroelution tube for the process to be carried out, so is more suited to self-packed or batch processed material rather than ready-packed colimuis. The antigen is eluted by the electric field into a small chamber sealed with a dialysis membrane and can be recovered in a small volume (400-600 JJ.1) of suitable buffer. The membrane enclosed chamber can be obtained with membranes of different molecular wei t cut-off to suit the size of the protein being eluted. 

Proteins were precipitated with ammonium sulphate (30%-50% ), desalted and loaded on Octyl-Sepharose CL-4B column (11 x 1.3 cm) equilibrated with the 0.03 M MBS buffer pH 6.0 with 120mM CaC. Unbound proteins were eluted with 0.01 M MBS pH 6.0 with 50 mM CaCl2. The PLD activity was found in the fractions eluted with 0.2 mM BDTA. Active fractions were pooled, concentrated on Centricon 30 and applied on the native PAGB (8%) according to Wang (3). Finally the active PLD protein was electroeluted from polyacrylamide gel. 

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