Reversible Negative Staining of Protein on Electrophoresis Gels
37
2.6
Fast Passive Elution of Proteins
Procedure (Castellanos-Serra et al. 1996, 1997)
Preparation of the crushing syringe (Heukeshoven and Dernick 1991): (Materials: 100 !-lm and 32 !-lm metal sieves can be purchased from F. Carl Schroetter,
Hamburg or any equivalent distributor. Cut two metal sieve disks (one of 100-!-lm
and one of 32-!-lm) to fit the internal diameter of a 1 mL (tuberculin) polypropylene syringe. Place the 32-!-lm disk at the bottom, followed by the 100 !-lm disk.
Press them tightly at the bottom with the aid of the syringe plunger. Flush the
syringe with lOX Laemmli running buffer several times, and next with MQW.
Protein elution:
1. Detect proteins by imidazole-SDS-zinc (2.2.3).
2. Cut out selected protein bands, chelate residual zinc and mobilize the protein
as described under 2.3.
3. Crush the gel band through the metal sieves by placing the gel band(s) in the
crushing syringe and forcing the gel through the sieves to obtain 32-!-lm gel
particles. Collect the gel slurry in a 1.5 mL microtube.
4. Flush the syringe with the selected elution buffer through the sieves (see
below) and collect the solution in the tube containing the gel slurry.
5. Incubate in Tris-Gly or ammonium bicarbonate 30-50 mM, pH 8.0 (it is not
necessary to add detergents) with moderate vortexing during 2 x 10 mins and
next, 1 x 1 min in water.
6. Collect the solution at each step by centrifuging for a few seconds. The elution
volume should be about twice the volume of the crushed gel at each elution
step. For highly hydrophobic proteins, substitute the second incubation in
Tris- glycine by an incubation in an aqueous solution containing 20 % formic
acid, 25 % isopropanol, 15 % acetonitrile.
Comments
i) Working with total radio-iodinated E .. coli proteins (radioactivity evenly distributed across all MW ranges) this procedure gave the following yield per
MW range: 95% (21-31 kDa), 94% (31-45 kDa) 92% (45-97 kDa), 91%
(higher than 97 kDa). These values are for proteins separated on a 12.5 % PA
gel, loaded without previous reduction, detected in 15 mins with ImH-SDSZinc and immediately eluted after the run.
ii) Same experiment as in (i) but proteins loaded in Laemmli buffer with mercaptoethanol give a significant drop in the yield for the higher MW range (only 65 %
recovered). When this gel was left for two days on the bench and then eluted, protein re-aggregation was observed and the recovery dropped to -27 % for the
high MW range. Therefore, to achieve high yields, proteins (fully alkylated in 8M
Urea, before electrophoresis, or loaded without previous reduction) should be
eluted from negatively stained or unstained gels immediately after the run.
iii) Generating microparticles of the gel is crucial for achieving a very rapid diffusion. For the same elution times, yields from 100-!-lm gel particles were
found lower than those from 32-!-lm particles.
37
2.6
Fast Passive Elution of Proteins
Procedure (Castellanos-Serra et al. 1996, 1997)
Preparation of the crushing syringe (Heukeshoven and Dernick 1991): (Materials: 100 !-lm and 32 !-lm metal sieves can be purchased from F. Carl Schroetter,
Hamburg or any equivalent distributor. Cut two metal sieve disks (one of 100-!-lm
and one of 32-!-lm) to fit the internal diameter of a 1 mL (tuberculin) polypropylene syringe. Place the 32-!-lm disk at the bottom, followed by the 100 !-lm disk.
Press them tightly at the bottom with the aid of the syringe plunger. Flush the
syringe with lOX Laemmli running buffer several times, and next with MQW.
Protein elution:
1. Detect proteins by imidazole-SDS-zinc (2.2.3).
2. Cut out selected protein bands, chelate residual zinc and mobilize the protein
as described under 2.3.
3. Crush the gel band through the metal sieves by placing the gel band(s) in the
crushing syringe and forcing the gel through the sieves to obtain 32-!-lm gel
particles. Collect the gel slurry in a 1.5 mL microtube.
4. Flush the syringe with the selected elution buffer through the sieves (see
below) and collect the solution in the tube containing the gel slurry.
5. Incubate in Tris-Gly or ammonium bicarbonate 30-50 mM, pH 8.0 (it is not
necessary to add detergents) with moderate vortexing during 2 x 10 mins and
next, 1 x 1 min in water.
6. Collect the solution at each step by centrifuging for a few seconds. The elution
volume should be about twice the volume of the crushed gel at each elution
step. For highly hydrophobic proteins, substitute the second incubation in
Tris- glycine by an incubation in an aqueous solution containing 20 % formic
acid, 25 % isopropanol, 15 % acetonitrile.
Comments
i) Working with total radio-iodinated E .. coli proteins (radioactivity evenly distributed across all MW ranges) this procedure gave the following yield per
MW range: 95% (21-31 kDa), 94% (31-45 kDa) 92% (45-97 kDa), 91%
(higher than 97 kDa). These values are for proteins separated on a 12.5 % PA
gel, loaded without previous reduction, detected in 15 mins with ImH-SDSZinc and immediately eluted after the run.
ii) Same experiment as in (i) but proteins loaded in Laemmli buffer with mercaptoethanol give a significant drop in the yield for the higher MW range (only 65 %
recovered). When this gel was left for two days on the bench and then eluted, protein re-aggregation was observed and the recovery dropped to -27 % for the
high MW range. Therefore, to achieve high yields, proteins (fully alkylated in 8M
Urea, before electrophoresis, or loaded without previous reduction) should be
eluted from negatively stained or unstained gels immediately after the run.
iii) Generating microparticles of the gel is crucial for achieving a very rapid diffusion. For the same elution times, yields from 100-!-lm gel particles were
found lower than those from 32-!-lm particles.