[ Care and Use ManUal ]
RAPIgeSt Sf Surfactant
I. IntroductIon
RapiGest™ SF is a reagent used to enhance in-gel (Ref. 1) and in-solu-
tion (Refs. 2-6) enzymatic digestions of proteins. Recent investigations
also support its use to solubilize glycoproteins prior to deglycosylation
with enzymes such as PNGase F (Publication in preparation). RapiGest
SF helps solubilize proteins making them more susceptible to enzy-
matic cleavage without significantly inhibiting enzyme activity. Unlike
other commonly used denaturants (e.g., SDS or Urea), RapiGest SF
does not modify peptides or suppress endoprotease activity. RapiGest
SF is compatible with enzymes such as Trypsin, Lys-C, Asp-N and Glu-
C and other enzymes. This reagent is easily removed after use allowing
MALDI-TOF MS, HPLC/MS, or HPLC/UV analyses of digested samples.
II. Storage and StabIlIty
The lyophilized powder is stable at room temperature until the expiration
date printed on the label (i.e., 3 years after packaging). Once reconsti-
tuted in high purity water or a buffer (pH 7–10) the solution is stable
for one week when stored at 2–8 °C. Long term storage of frozen ali-
quots is possible but not recommended due to potential solubilization
issues of RapiGest SF or storage buffer.
contentS
I. IntroductIon
II. Storage and StabIlIty
III. reconStItutIon of RAPIgeSt Sf powder
IV. SuggeSted procedure for In-SolutIon dIgeStIonS
V. SuggeSted procedure for In-gel dIgeStIonS
VI. SuggeStIonS for worKIng wItH proteolytIc reSIStant
or HydropHobIc proteInS
VII. SuggeSted SaMple preparatIon for Hplc and MaSS
SpectroMetry
Note: RapiGest SF hydrolyzes in acidic solutions (half life 8 min. at pH
2 and 60 min. at pH 3).
III. reconStItutIon of rapIgeSt Sf powder
Shown in Table 1, are the volumes required to reconstitute the 1 mg
RapiGest SF powder in water or buffer to obtain preferred concentra-
tions. If Mass Spec analysis is required, the recommended buffer is
50mM Ammonium Bicarbonate (NH4HCO3). Alternative buffers, such
as 10 mM Tris-HCl or 25 mM sodium phosphate, are also RapiGest SF
compatible.
RapiGest SF Surfactant 1
[ Care and Use ManUal ]
Table 1: Reconstitution of RapiGest SF Powder
Volume of Buffer Added
to RapiGest SF Vial
1 ml 0.1%
500 µL 0.2%
200 µL 0.5%
100 µL 1%
50 µL 2%
The recommended concentration is 0.1% (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).
RapiGest SF
Concentration (w/v)
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.)
1. Suspend the 1 mg of lyophilized RapiGest SF powder in 1 mL of
50 mM Ammonium Bicarbonate (NH4HCO3) to give 0.1% (w/v).
2. Suspend protein pellet in the 0.1% RapiGest SF solution and
vortex.
3. Add DTT to the protein sample to a final concentration of 5 mM.
2. Slice gel spots into <1 mm3 cubes, transfer the gel pieces into a
1.5 ml microcentrifuge tube, add 30 μl of H2O and leave at room
temperature for 15 min.
3. Remove the supernatant, add 20 μl of 50% acetonitrile, mix, and
leave for 15 min.
4. Remove the supernatant, add 30 μl of 100% acetonitrile, mix,
and leave for 15 min.
5. Remove the supernatant, add 20 μl of 0.1 M NH4HCO3, mix, and
leave for 5 min.
6. Add 30 μl of 100% acetonitrile, mix, and leave for 15 min.
7. Remove the supernatant and completely dry the gel pieces with a
Speed Vac.
8. Add 50 μl of 10 mM DTT in 0.1 M NH4HCO3 and incubate at
56 °C for 45 min.
9. Remove the supernatant, add 30 μl of 55 mM iodoacetamide in
0.1 M NH4HCO3 and leave in the dark for 30 min.
(Repeat steps 3–7)
10. Add 20 μl of 0.1% RapiGest SF solution in 50 mM NH4HCO3 and
incubate at 37 °C for 10 min.
11. Remove excess solution and completely dry the gel pieces with a
Speed Vac.
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
15 mM and place the sample in the dark for 30 minutes.
7. Add enzyme for digestion (1:100 to 1:20, w/w).
8. Incubate the samples at 37 °C for (1 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.)
1. Excise protein spots from gel, wash with 30 μl of H2O and leave
for 15 min.
RapiGest SF Surfactant 2
12. Gradually (single drop at a time) add trypsin (12.5 ng per μL in
50 mM NH4HCO3) to the gel slice until the gel is reswollen.
Continue to incubate on ice for 45 min.
13. Remove excess solution, add 20 μl of 50 mM NH4HCO3 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 ~ 100 °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.
[ Care and Use ManUal ]
VII. SuggeSted SaMple preparatIon for Hplc and MaSS
SpectroMetry
1. Add TFA to the digested protein samples. T he final TFA concen-
tration should be approx. 0.5% (pH < 2).
(Note: Obtain high purity Trifluoroacetic acid (TFA). The purchase and use
of sealed vials containing 1 mL of high purity TFA is recommended.)
2. Incubate the sample at 37 °C for 30 – 45 minutes. Slight cloudi-
ness should be observed.
3. Centrifuge acid treated samples at 13,000 rpm for 10 minutes.
The hydrolytic RapiGest SF by-products are water immiscible
therefore, some precipitation may be observed.
4. Carefully transfer the solution to another microcentrifuge tube or
HPLC sample vial.
5. Proceed with MALDI Tof, HPLC or LC/MS analysis.
RapiGest SF Surfactant 3
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©2007 Waters Corporation. Waters Corporation, Waters, The
Science of What’s Possible, and RapiGest are trademarks of Waters
Corporation.
July 2007 715000122 Rev E EY-PDF
RapiGest SF Surfactant 4
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com
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