Proteomics Protocols
Materials:
Note: Make all materials fresh!
25 mM Ammonium Bicarbonate (FW 79.06)
Dilute 98 mg ammonium bicarbonate in 50 ml water (1.9 mg/ml)
8M Urea:
Dilute 0.96 gram of urea in 2.0 mL of 25 mM ammonium bicarbonate solution (480 mg/ml)
Reducing reagent (make fresh just before needed):
Dissolve 30 mg of DTT in 0.20 mL of 25 mM ammonium bicarbonate solution to make 1M DTT
Alkylating reagent (make fresh just before needed):
Dissolve 36 mg (0.036 g) in 1 mL of ammonium bicarbonate solution to make 200 mM iodoacetamide.
0.1 ug /ul Trypsin solution
10 ug of trypsin in 100 uL of bicarbonate buffer (or water, or provided solution).
Trypsin can be aliquoted into 10ul tubes and frozen at -20. Make fresh or thaw an aliquot just prior to use.
Digestion Procedure
1. Reconstitute sample in approximately 20 uL of 8.0 M urea
2. Add 1 uL of Reducing Reagent and mix the sample by gentle vortex
3 Reduce the mixture for 1 hour at room temperature or in an oven at 37 C
4. Add 20 uL of Alkylating Reagent and alkylate for 1 hour at room temperature in the dark and with shaking.
5. Add 4 uL of Reducing Reagent to consume any leftover alkylating agent
6. Add 120 uL of ammmonium bicarbonate solution to dilute the urea
7. Add trypsin in appropriate ratio (1:30) to approximate amount of protein by weight. Digest overnight at 37 C.
8. In the morning, add 1 ul of formic acid to the sample and gently vortex.
9. Perform a clean up step as directed by your mass spectrometry facility.
Note: Following digestion, the sample can be frozen at -20 or -80 if it will not be used immediately for mass
spectrometry.
Back to top of page
Protocol I - to be used when detergents may interfere with mass spectrometry.
1. Perform immunoprecipitation (IP) according to your protocol
2. After final wash of beads, wash the beads 2 times with 300 ul Tris HCl pH 8.0.
3. Add 50 ul 0.1 M glycine pH 2.5 (this solution should not have to be pH'd)
a. Alternatively, 0.1 M Citric Acid, pH 2.2 can be used.
4. Centrifuge as previously for IP and collect supernatant in tube containing 5 ul 1M Tris base to neutralize.
5. Repeat step 3 two more times, collecting and combining the supernatants each time. Your total volume of
supernatant should be 150 ul and your total volume of 1M tris base should be 15 ul.
6. Speed vac or lyophilize the eluant and perform an in-solution digest.
7. Add 40 ul 1x Laemmli Sample buffer to the beads and run a 1D gel if desired. This will determine the
efficiency of elution.
Protocol II - to be used when detergents will NOT interfere with mass spectrometry.
1. Perform immunoprecipitation (IP) according to your protocol.
2. After final wash of beads, wash the beads 2 times with 300 ul Tris HCl pH 8.0.
3. Add 50 ul 0.1 M glycine pH 2.5 (this solution should not have to be pH'd).
a. Alternatively, 0.1 M Citric Acid, pH 2.2 can be used.
4. Centrifuge as previously for IP and collect supernatant in tube containing 5 ul 1M Tris base to neutralize.
5. Repeat step 3 two more times, collecting and combining the supernatants each time.
Your total volume of supernatant should be 150 ul and your total volume of 1M tris base should be 15 ul.
6. Speed vac or lyophilize the eluant and perform an in-solution digest
7. Add 40 ul 1x Laemmli Sample buffer to the beads and run a 1D gel if desired. This will determine the efficiency of elution.
Notes:
1. Avoid pipetting beads with supernatant. You may have to increase the speed of your spin, and/or use gel tips.
2. Avoid KERATIN! Remember that the next step is digestion of your proteins. If any keratin is present in your
sample, it will likely appear as the number one protein in subsequent steps. Unless your goal is to characterize
keratin, please take precautions and make certain you review our information on a Keratin-Free Environment.
3. As an alternative to citric acid or glycine, you may want to try using formic acid/H2O/Acetonitrile in 1:20:20 ratio.
4. In order to test the efficiency of your elution protocol, we recommend running the experiment in duplicate.
For sample 1, follow a protocol outlined above, but do NOT pool supernatants. To each supernatant, and to the
remaining pellet, add Laemmli buffer. For sample 2, resuspend the pellet in 40ul Laemmli. Run all on a 1D gel.
You may need to adjust the volumes.
Back to top of page
