Elution of lipopolysaccharides from polyacrylamide gels.

A simple procedure for elution in water of bacterial lipopolysaccharides (LPS) from sodium dodecyl sulfate-polyacrylamide gels is described. It consists of the combination of three principal steps: first, highly sensitive on-gel LPS detection (1-10 ng/band) with zinc-imidazole (negative or reverse staining); second, washing of the individual LPS band in a solution of a zinc-complexing agent (e.g., 100 mM EDTA); and finally, elution of the LPS (100-200 microliters water for a 0.5-microgram LPS band) from gel microparticles for 3 h at room temperature. Using this procedure, we have successfully eluted a variety of LPS forms from Bordetella pertussis, Escherichia coli 0111:B4, E. coli K-235, Salmonella enteritidis, and Pseudomonas aeruginosa. Elution recovery of rough or semismooth LPS was about 70-80%, while that of smooth LPS was only about 10%. Eluted LPS was biologically active as tested by limulus amebocyte lysate and TNF-alpha assays.

High yield elution of proteins from sodium dodecyl sulfate-polyacrylamide gels at the low-picomole level. Application to N-terminal sequencing of a scarce protein and to in-solution biological activity analysis of on-gel renatured proteins.

A simple, reliable procedure for practically quantitative (90-98%) and fast (< 30 min) elution of proteins from SDS-PA gels is described with reproducible recoveries in the range from 100 to 1 pmol per band, which does not require the inclusion of detergents in the elution buffer. It consists in the combination of (1) highly sensitive on-gel protein detection (50 mol per band) with imidazole-SDS-zinc (reverse staining), (2) crushing of the protein band to produce 32-micron gel particles, and (3) vortexing of the slurry in a solution of a zinc-complexing agent, e.g. glycine 0.5 M or EDTA 100 mM (100 microliters for a 100-pmol BSA band), at room temperature. Eluted proteins can be directly analyzed by RP-HPLC, quantitatively loaded onto a PVDF membrane, or, provided that they are previously renatured on-gel, analyzed by biological activity tests. The application of the procedure to in-solution enrichment of scarce proteins for N-terminal analysis is shown.

A procedure for protein elution from reverse-stained polyarcylamide gels applicable at the low picomole level: An alternative route to the preparation of low abundance proteins for microanalysis.

We developed a technique that allows rapid protein elution from polyacrylamide gel bands at room temperature into a detergent-free buffer (elution time 2 x 10 min, total working time about 30 min) with high yields (90-98%) even at a low picomole level (1 picomole per band). Its efficacy relies on the combination of protein detection by reverse staining with the enhancement of protein diffusion after gel crushing. Detection is accomplished by gel incubation in an imidazole solution, followed by incubation in a zinc salt solution to develop a negative stain pattern. Proteins are eluted by zinc complexation in Laemmli electrophoresis buffer (Tris + glycine), from which sodium dodecyl sulfate is omitted to allow direct subsequent microanalysis, e.g. high performance liquid chromatography (HPLC) and automatic sequencing. A variety of proteins were eluted efficiently (with no apparent restriction due to their intrinsic properties) as quantified with radioiodinated total E. coli proteins. Yields were independent of acrylamide concentration, protein molecular mass (from 10 to 100 kDa) and the amount (from 1 to 100 picomole) of protein in the band. This protocol was derived from a quantitative evaluation of the effect of protein staining and of sample reduction prior to electrophoresis on elution yields. For N-terminal sequencing, the protein eluate was automatically loaded on a polyvinylidene difluoride (PVDF) membrane with conventional HPLC equipment; both loading and membrane clean-up were monitored at 206 nm. By simultaneously processing several analytical bands, the procedure allowed trace enrichment of a natural scarce protein that was N-terminal sequenced.

Expression and folding of an interleukin-2-proinsulin fusion protein and its conversion into insulin by a single step enzymatic removal of the C-peptide and the N-terminal fused sequence.

We report the expression in E. coli of a proinsulin fusion protein carrying a modified interleukin-2 N-terminal peptide linked to the N-terminus of proinsulin by a lysine residue. The key aspects investigated were: (a) the expression of the fused IL2-PI gene, (b) the folding efficiency of the insulin precursor when still carrying the N-fused peptide and (c) the selectivity of the enzymatic cleavage reaction with trypsin in order to remove simultaneously the C-peptide and the N-terminal extension. It was found that this construction expresses the chimeric proinsulin at high level (20%) as inclusion bodies; the fused protein was refolded at 100-200 micrograms/ml to yield about 80% of correctly folded proinsulin and then it was converted into insulin by prolonged reaction (5 h) with trypsin and carboxypeptidase B at a low enzyme/substrate rate (1:600). This approach is based on a single enzymatic reaction for the removal of both the N-terminal fused peptide and the C-peptide and avoids the use of toxic cyanogen bromide.

Protein reverse staining: high-efficiency microanalysis of unmodified proteins detected on electrophoresis gels.

A methodology is presented for efficiently gaining structural information from electrophoresed proteins after on-gel detection by imidazole-sodium dodecyl sulfate-zinc reverse staining. As a consequence of reverse staining, (a) protein bands arise transparent against a deep white-stained background, limits of detection being in the femtomole range; (b) there is no loss of image when the gel is kept in distilled water (even during years); and (c) protein bands result immobilized, i.e., they do not diffuse upon gel storage. To recover reverse-stained proteins or fragments thereof from the gel, the immobilization of bands must first be abrogated by chelating the zinc ions from stain (protein mobilization). We had originally described mobilization at low pH by using citric acid. Here, we improve this procedure regarding the protein electrotransfer. We demonstrate that mobilization is efficiently done at neutral to alkaline pH by short-term (5 to 10 min) incubation of the gel in a buffer containing glycine or dithiothreitol prior to transfer. Moreover, mobilization was most simply performed by just adding the zinc chelating agent to the transfer buffer. Reverse staining and the new mobilization procedure made electrotransferring single protein bands from gel onto small-sized (13 x 5 mm2) PVDF membrane pieces in mini sandwich-like assemblies practical. Equipment is described for the protein electroblotting in such minisandwiches. Microsequence analysis of the electroblotted proteins showed initial yields in the range of those achieved when the transfer was done from unstained control gels. Protein bands kept in the reverse-stained gel for prolonged time periods (even for as long as 2 years) could be similarly analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)