Reversible Negative Staining of Protein on Electrophoresis Gels
41
Recovery of Peptides: (At each step elution volumes are about twice the volume
of crushed gel. Do not store solutions containing organic solvents in polypropylene vials as they may release polymers that can interfere with analysis by mass
spectrometry.) Centrifuge the gel for a few seconds and collect the supernatant.
Add approximately two-fold of the crushed gel volume of digestion buffer, vortex
for 10 mins, centrifuge and collect the solution. Add water to the gel slurry, vortex briefly and collect the solution after centrifugation. Then, add 50 % acetonitrile, 0.1 % TFA, vortex for 10 mins. Collect the solution. Repeat this step. If
highly hydrophobic peptides are suspected to be present, replace the second
extraction with 50 % acetonitrile by an extraction for 10 mins. with an aqueous
solution containing 20 % formic acid, 15 % isopropanol, 25 % acetonitrile. Finally,
add 90 % acetonitrile and vortex vigurously for 2 mins. centrifuge and collect the
solution. Concentrate the solution by centrifugal evaporation.
Approximate Time Schedule for the Protocol (in mins):
Staining
15
Destaining and removal ofSDS
16
Gel crushing
10
Digestion
240
Peptide elution
20-30
Starting with a ready to use mini-gel, this time schedule allows separation, detection, digestion and extraction in one working day.
Comments
i) During gel staining with Im-SDS-Zinc, a minor but significant amount of
SDS is incorporated onto the proteins in the gel. In the present protocol, SDS
is removed before digestion by incubating the slice in 30 % ACN. During this
step, Zn II is also removed by incorporating glycine to the ACN solution. If
several bands are processed simultaneously, the volume of 30 % ACN should
be scaled up proportionally, to assure complete removal of SDS.
ii) Using total radioiodinated E- coli proteins separated on a 12.5 % polyacrylamide gel, losses from the gel lane during zinc chelation and removal of SDS
in 30 % ACN (step 2) were determined to be only 0.5 -1 %.
iii) For micro digestion, proteins are either loaded in Laemmli sample buffer
without reducing agent, fully reduced and alkylated in urea solution before
loading to the gel as described above or pyridylethylated in the gel band
(Moritz et al, 1996). An incomplete reduction/alkylation can lead to in-gel reaggregation of proteins which, in turn, affect the efficiency in digestion and
may generate peptides difficult to identify, therefore, special care should be
taken during this step to assure full derivatization of cysteine residues.
Advantages
The advantages of this protocol in comparison to the established ones for Coomassie blue-detected proteins are derived from the constant pH during separation, detection and digestion, that avoids protein precipitation in the gel and the
possibility of protein modification by stain related chemicals, and from the high
mass exchange between gel microparticles and the solution. Consequently, it
41
Recovery of Peptides: (At each step elution volumes are about twice the volume
of crushed gel. Do not store solutions containing organic solvents in polypropylene vials as they may release polymers that can interfere with analysis by mass
spectrometry.) Centrifuge the gel for a few seconds and collect the supernatant.
Add approximately two-fold of the crushed gel volume of digestion buffer, vortex
for 10 mins, centrifuge and collect the solution. Add water to the gel slurry, vortex briefly and collect the solution after centrifugation. Then, add 50 % acetonitrile, 0.1 % TFA, vortex for 10 mins. Collect the solution. Repeat this step. If
highly hydrophobic peptides are suspected to be present, replace the second
extraction with 50 % acetonitrile by an extraction for 10 mins. with an aqueous
solution containing 20 % formic acid, 15 % isopropanol, 25 % acetonitrile. Finally,
add 90 % acetonitrile and vortex vigurously for 2 mins. centrifuge and collect the
solution. Concentrate the solution by centrifugal evaporation.
Approximate Time Schedule for the Protocol (in mins):
Staining
15
Destaining and removal ofSDS
16
Gel crushing
10
Digestion
240
Peptide elution
20-30
Starting with a ready to use mini-gel, this time schedule allows separation, detection, digestion and extraction in one working day.
Comments
i) During gel staining with Im-SDS-Zinc, a minor but significant amount of
SDS is incorporated onto the proteins in the gel. In the present protocol, SDS
is removed before digestion by incubating the slice in 30 % ACN. During this
step, Zn II is also removed by incorporating glycine to the ACN solution. If
several bands are processed simultaneously, the volume of 30 % ACN should
be scaled up proportionally, to assure complete removal of SDS.
ii) Using total radioiodinated E- coli proteins separated on a 12.5 % polyacrylamide gel, losses from the gel lane during zinc chelation and removal of SDS
in 30 % ACN (step 2) were determined to be only 0.5 -1 %.
iii) For micro digestion, proteins are either loaded in Laemmli sample buffer
without reducing agent, fully reduced and alkylated in urea solution before
loading to the gel as described above or pyridylethylated in the gel band
(Moritz et al, 1996). An incomplete reduction/alkylation can lead to in-gel reaggregation of proteins which, in turn, affect the efficiency in digestion and
may generate peptides difficult to identify, therefore, special care should be
taken during this step to assure full derivatization of cysteine residues.
Advantages
The advantages of this protocol in comparison to the established ones for Coomassie blue-detected proteins are derived from the constant pH during separation, detection and digestion, that avoids protein precipitation in the gel and the
possibility of protein modification by stain related chemicals, and from the high
mass exchange between gel microparticles and the solution. Consequently, it