40
2.8
Micro-Deglycosylation
Procedure
L. CASTELLANOS-SERRA et aI.
Band conditioning: Immediately after electrophoresis, cut the band of interest.
Incubate the band in 50 mM sodium phosphate, 0.3 M glycine, pH 7.5, 2 x 5 mins;
and next in water, 2 x 5 mins. Deglycosylation and analysis: Crush the gel bands
to yield 32 [tm particles and collect the gel slurry. To 60 [tl of the digestion buffer
(10 mM sodium phosphate, 20 % glycerol, 1 % NP40) add 7.5 [tl of a 1:10 dilution
of PNGase F (BioLabs). This solution is added to the crushing syringe and
flushed through the sieves to collect any gel particle remaining in the syringe
Vortex for 10 mins. and digest for 2-3 h at 37°C. Elute the protein as described
in 2.6. Relevant information can then be obtained by comparing the electrophoretic profile of the glycosylated and deglycosylated protein both on SDS- and IEFslab gels.
Advantages
This is a very rapid protocol for in-gel deglycosylation of proteins. The reaction
is generally complete after 2 h; due to the combination of generating gel microparticles after a fully reversible fIxative negative staining, and the use of the
recombinant PNGAse F (BioLabs).
2.9
Microdigestion of Reverse Stained Proteins (Castellanos-Serra et 01. 7999)
Immediately after electrophoresis, cut the bands of interest. Perform simple zinc
chelation (see Section 2.3.1) by incubating for 2 x 8 mins. in 1 mL 50 mM Tris,
200 mM glycine, pH 8.3 containing 30 % acetonitrile. Wash briefly (2 x 1 min)
with 1 mL 50 mM Tris, pH 8.3. Alternatively, perform in-gel alkylation (see Section 2.3.2) by incubating for 1 x 5 mins and 1 X 30 mins in 25 mM Tris, 50 mM
glycine pH 8.0 containing 0.01 M DTT. To S-pyridylethylate, add 4-vinylpyridine
(2 % v/v) in the reduction buffer containing the gel and incubate in the dark, at
25°C for 60 mins. After incubation, halt the alkylation by adding I3-Mercaptoethanol (2 % v/v) then discard the solution and wash the gel band extensively in
water.
Digestion: Crush the gel band through a crushing syringe provided with metal
sieves of 100 and 32 [tm (see 2.6), to yield 32-[tm gel particles. Collect the gel
slurry. To 30-60 [tl of the digestion buffer (see below), add 0.3-0.5 [tg of a proteolytic enzyme. This solution is added to the crushing syringe and flushed through
the sieves to collect any gel particles remaining in the syringe. The volume of
solution should be just enough to cover the gel slurry. Vortex for 10 mins and
then incubate at 37°C for four-fIve h. Check during the incubation that the solution remains to cover the gel slurry. If necessary add more buffer.
Recommended Digestion Buffers: Trypsin: 50 mM NH4C03; Glu-C endoproteinase: 25 mM NH4C03, pH 7.8; Asp-N endoproteinase: 50 mM phosphate, pH 8.0;
Lys-C endoproteinase: 50 mM Tris, pH 9.3.
2.8
Micro-Deglycosylation
Procedure
L. CASTELLANOS-SERRA et aI.
Band conditioning: Immediately after electrophoresis, cut the band of interest.
Incubate the band in 50 mM sodium phosphate, 0.3 M glycine, pH 7.5, 2 x 5 mins;
and next in water, 2 x 5 mins. Deglycosylation and analysis: Crush the gel bands
to yield 32 [tm particles and collect the gel slurry. To 60 [tl of the digestion buffer
(10 mM sodium phosphate, 20 % glycerol, 1 % NP40) add 7.5 [tl of a 1:10 dilution
of PNGase F (BioLabs). This solution is added to the crushing syringe and
flushed through the sieves to collect any gel particle remaining in the syringe
Vortex for 10 mins. and digest for 2-3 h at 37°C. Elute the protein as described
in 2.6. Relevant information can then be obtained by comparing the electrophoretic profile of the glycosylated and deglycosylated protein both on SDS- and IEFslab gels.
Advantages
This is a very rapid protocol for in-gel deglycosylation of proteins. The reaction
is generally complete after 2 h; due to the combination of generating gel microparticles after a fully reversible fIxative negative staining, and the use of the
recombinant PNGAse F (BioLabs).
2.9
Microdigestion of Reverse Stained Proteins (Castellanos-Serra et 01. 7999)
Immediately after electrophoresis, cut the bands of interest. Perform simple zinc
chelation (see Section 2.3.1) by incubating for 2 x 8 mins. in 1 mL 50 mM Tris,
200 mM glycine, pH 8.3 containing 30 % acetonitrile. Wash briefly (2 x 1 min)
with 1 mL 50 mM Tris, pH 8.3. Alternatively, perform in-gel alkylation (see Section 2.3.2) by incubating for 1 x 5 mins and 1 X 30 mins in 25 mM Tris, 50 mM
glycine pH 8.0 containing 0.01 M DTT. To S-pyridylethylate, add 4-vinylpyridine
(2 % v/v) in the reduction buffer containing the gel and incubate in the dark, at
25°C for 60 mins. After incubation, halt the alkylation by adding I3-Mercaptoethanol (2 % v/v) then discard the solution and wash the gel band extensively in
water.
Digestion: Crush the gel band through a crushing syringe provided with metal
sieves of 100 and 32 [tm (see 2.6), to yield 32-[tm gel particles. Collect the gel
slurry. To 30-60 [tl of the digestion buffer (see below), add 0.3-0.5 [tg of a proteolytic enzyme. This solution is added to the crushing syringe and flushed through
the sieves to collect any gel particles remaining in the syringe. The volume of
solution should be just enough to cover the gel slurry. Vortex for 10 mins and
then incubate at 37°C for four-fIve h. Check during the incubation that the solution remains to cover the gel slurry. If necessary add more buffer.
Recommended Digestion Buffers: Trypsin: 50 mM NH4C03; Glu-C endoproteinase: 25 mM NH4C03, pH 7.8; Asp-N endoproteinase: 50 mM phosphate, pH 8.0;
Lys-C endoproteinase: 50 mM Tris, pH 9.3.