Waters RapiGest SF Surfactant - Manual (Page 2)

[ Care and Use ManUal ]
RapiGest SF Surfactant 2
2. Slice gel spots into < mm 3 cubes, transfer the gel pieces into a
.5 ml microcentrifuge tube, add 30 μl of H
2
O and leave at room
temperature for 5 min.
3. Remove the supernatant, add 20 μl of 50% acetonitrile, mix, and
leave for 5 min.
4. Remove the supernatant, add 30 μl of 00% acetonitrile, mix,
and leave for 5 min.
5. Remove the supernatant, add 20 μl of 0. M NH
4
HCO
3
, mix, and
leave for 5 min.
6. Add 30 μl of 00% acetonitrile, mix, and leave for 5 min.
7. Remove the supernatant and completely dry the gel pieces with a
Speed Vac.
8. Add 50 μl of 0 mM DTT in 0. M NH
4
HCO
3
and incubate at
56 °C for 45 min.
9. Remove the supernatant, add 30 μl of 55 mM iodoacetamide in
0. M NH
4
HCO
3
and leave in the dark for 30 min.
(Repeat steps 3–7)
0. Add 20 μl of 0.% RapiGest SF solution in 50 mM NH
4
HCO
3
and
incubate at 37 °C for 0 min.
. Remove excess solution and completely dry the gel pieces with a
Speed Vac.
2. Gradually (single drop at a time) add trypsin (2.5 ng per μL in
50 mM NH
4
HCO
3
) to the gel slice until the gel is reswollen.
Continue to incubate on ice for 45 min.
3. Remove excess solution, add 20 μl of 50 mM NH
4
HCO
3
and
incubate at 37 °C overnight.
VI. SuggeStIonS for worKIng wItH proteolytIc reSIStant
or HydropHobIc proteInS
When dealing with hydrophobic proteins such as membrane proteins,
the following modifications are recommended: (See Ref. 3 for more
information)
• Boil the protein/ RapiGest SF mixture at ~ 00 °C for 5 minutes,
cool the sample down before adding proteolytic enzymes.
• Proteins that are highly resistant to protease may require longer
digestion times or higher RapiGest SF concentrations.
Table 1: Reconstitution of RapiGest SF Powder
The recommended concentration is 0.% (w/v) RapiGest SF. Hydro-
phobic proteins may require higher RapiGest SF concentrations (see
note for recommendations for working with proteolytic resistant or
hydrophobic proteins).
IV. SuggeSted procedure for In-SolutIon dIgeStIonS
(Note: Modifications to this suggested method, for example increased
digestion times, may be necessary depending upon the target
proteins.)
. Suspend the mg of lyophilized RapiGest SF powder in mL of
50 mM Ammonium Bicarbonate (NH
4
HCO
3
) to give 0.% (w/v).
2. Suspend protein pellet in the 0.% RapiGest SF solution and
vortex.
3. Add DTT to the protein sample to a final concentration of 5 mM.
4. Heat the sample at 60 °C for 30 minutes.
5. Cool the sample to room temperature.
6. Add Iodoacetamide to the sample to a final concentration of
5 mM and place the sample in the dark for 30 minutes.
7. Add enzyme for digestion (:00 to :20, w/w).
8. Incubate the samples at 37 °C for ( hr to overnight depending
upon protein hydrophobicity) for optimum enzymatic digestion.
V. SuggeSted procedure for In-gel dIgeStIonS
(Note: Modifications to this suggested method may be necessary
depending upon the target proteins and applications.)
. Excise protein spots from gel, wash with 30 μl of H
2
O and leave
for 5 min.
Volume of Buffer Added
to RapiGest SF Vial
RapiGest SF
Concentration (w/v)
ml 0.%
500 µL 0.2%
200 µL 0.5%
00 µL %
50 µL 2%