In-Gel Digestion
Notes:
- Always wear gloves and change them often
- Use MilliQ water or HPLC Grade water to
making all solutions
- Preferable to work in a clean hood. If
unable to work in clean, protected area, wear a solid hair net and
mask to avoid contamination of the samples.
- Incubations are at room temperature unless
otherwise noted.
- Always centrifuge sample prior to removing
supernatant.
- Larger bands will require more solution
volume.
- It is very important to use siliconized
tubes to prevent sample loss.
- It is preferable to have a positive control
(i.e. known protein) if at all possible.
- Always have a negative control.
Chemical Reagents:
- Trypsin: Promega sequencing grade
modified trypsin (V511A), 20 mg lyophilized powder, can be stored
in solution for several weeks at -20°C.
- Endoproteinase ASP-N:
SIGMA (P 3303).
- Glutamic Acid-C: PrincetonSeparations
Sequencing grade modified Glutamic Acid-C (EN-140).
- ddH2O: MilliQ water or
HPLC water.
Solutions:
1 M Dithiothreitol (DTT)
- 154.3 mg of DTT +1000 mL ddH2O
1 M Ammonium Bicarbonate (NH4HCO3)
- 79.06 mg of NH4HCO3 +
1000 mL ddH2O
50% Acetonitrile in ddH2O:
- 250 mL of acetonitrile + 250 mL of ddH2O
100 mM NH4HCO3:
- 100 mL of 1 M NH4HCO3 in 900 mL of ddH2O
10 mM DTT in 100 mM NH4HCO3:
- 10 mL of 1 M DTT + 890 mL ddH2O+
100 mL 1 M NH4HCO3
55 mM iodoacetamide in 100 mM NH4HCO3:
- 10 mg of iodoacetamide + 900 mL ddH2O
+ 100 mL 1 M NH4HCO3
1 M Tris-Cl
- 121.1 g Tris base in 800 mL of ddH2O.
Adjust the pH to the desired value by adding concentrated HCl.
Trypsin digestion solution
- 25 mM NH4HCO3 + 2.5
mM CaCl2 with 12.5 ng/mL of Trypsin
- 2.5 mL of 1 M CaCl2 + 25 mL of 1
M NH4HCO3 + 972.5 mL of ddH2O
- Take100 mL of above solution add 1.25 mL of
1 mg/mL Trypsin solution (Trypsin usually prepared in 100 mM NH4HCO3)
25 mM NH4HCO3
- 25 mL of 1 M NH4HCO3+
975 mL of ddH2O
ASP-N digestion solution
- 100 mM NH4HCO3 (or
100mM Tris-HCl pH 8.5) with 12.5 ng/mL of ASP-N.
Glutamic Acid-C digestion solution
- 50 mM NH4HCO3 (or
50mM Tris-HCl pH 8.0) with 12.5 ng/mL of Glutamic Acid-C.
10 mM of DTT in ddH2O
- 10 mLof 1 M DTT + 995 mL of ddH2O
5% formic acid
- 57 mL of stock formic acid (88%) + 943 mL
of ddH2O
0.1% Trifluoroacetic (TFA)
- 1 mL of TFA + 999 mL of ddH2O
In-Gel Digestion for
Coomassie Stained Gels
1. Excision protein bands or
spot from gels
|
a |
Rinse the gel with 2 aliquots of water,
shaking for 10min each. |
| b |
Transfer the gel into a suitable size tissue
culture dish. |
| c |
Excise bands of interest with clean scalpel,
cutting as close to the edge of the band as possible. It is
important to reduce the volume of background gel. |
| d |
Cut the same size gel piece from a blank
region (i.e. no protein) of the gel for a negative control.
|
| e |
Chop the excised bands into cubes (about
1x1x1 mm). Transfer gel particles into a siliconized tube (0.5mL
Eppendorf). |
2. Washing (With occasional vortexing, then centrifuge
for seconds before removing the liquid)
|
a |
To each of the chopped up bands, add 100 mL
of ddH2O and incubate for 15 min. |
| b |
Discard ddH2O, then add 40 mL of
50/50 acetonitrile / ddH2O and incubate 15
minutes (Repeat this step 2 to 3 times; some strongly stained
bands will still be light blue). |
| c |
Discard solution, then add 40 mL of
acetonitrile. Incubate until gel pieces are white and sticky
(approx. 5 min.) |
| d |
Discard solution, then add 40 mL of 100 mM NH4HCO3.
Incubate for 5 minutes to rehydrate the gel pieces. |
| e |
Add 40 mL of acetonitrile to make 1:1 solution of
acetonitrile:100 mM NH4HCO3. Incubate 15 minutes. |
| f |
Discard solution, then dry samples in a
speedvac until completely dry. |
| g |
Gel pieces can be stored at 4°C or -20°C for
overnight. |
3. Reduction and Alkylation
|
a |
Remove samples from the speedvac and let it
cool. |
| b |
To each tube add 40 mL of 10 mM DTT/100 mM NH4HCO3
(For cysteine rich protein, we suggest to use 50 mM DTT
instead) then incubate in a water bath at 56°C for 45
minutes. |
| c |
Remove samples from bath and let cool. |
| d |
Discard solution and immediately add 40 mL of
55 mM iodoacetamide/100 mM NH4HCO3 (For
cysteine rich protein, we suggest to use 200 mM iodoacetamide
instead), then incubate at room temperature for 30
minutes in the dark. |
| e |
At this point, the stain is totally removed
and the gel should be clear. |
| f |
Discard solution, then wash with 100 mL of
100 mM NH4HCO3 and incubate for 5 minutes. |
| g |
Discard solution, then add 100 mL 50%
acetonitrile in
water and incubate for 15 minutes. |
| h |
Discard solution, then add 40 mL of
acetonitrile. Incubate until gel pieces are white and sticky. (2
to 3 minutes) |
| i |
Discard solution, then add 40 mL of 100 mM NH4HCO3.
Incubate 5 minutes to rehydrate the gel pieces. |
| j |
Add 40 mL of acetonitrile to make 1:1 solution, then
incubate 15 minutes. |
| k |
Discard solution, then dry samples in a speed
vacuum until completely dry. |
| l |
The gel can store at 4°C overnight or -20°C
until digestion occurs. |
4. Digestion
|
a |
Add 10-20 mL (enough to cover pieces) of the
appropriate enzyme solution (see Table 1,
and Table 2), then incubate for 45 min at 4°C
(ice bath). Add more solution if the pieces absorb all of the
liquid.
Table 1
Protein quantity
|
>1ug protein |
100-1000ng protein
|
< 100ng protein
|
Enzyme (ng/ uL)
|
25
|
12.5
|
6.25 |
Digestion solution without enzyme (uL)
|
10 |
10 |
10 |
|
| b |
Remove excess solution and discard. Add
20-30mL of same enzyme digestion buffer without enzyme (enough
to cover gel pieces), then incubate overnight at suitable
temperature (see Table 2).
Table 2
| Enzyme |
Company |
Digestion Buffer
|
Digestion temperature
|
Sequencing grade modified trypsin
|
Promega
|
25 mM NH4HCO3
and 2.5mM CaCl2, pH 7.8
|
37°C
|
Endoproteinase ASP-N
|
SIGMA
|
100 mM NH4HCO3
(or 100mM Tris-HCl ) pH 8.5
|
37°C
|
Sequencing grade modified Glutamic
Acid-C
|
Princeton Separations, Inc.
|
50 mM NH4HCO3
(or 50mM Tris-HCl ) pH 8.0
|
30°C
|
|
5. Extraction of peptides from gel
|
a |
The next morning (after about 16-17 hours of
digestion), sonicate the tubes for 5 min in cool water. Remove
supernatant and save.
|
| b |
To the gel in tubes add 20-30 mL of 25 mM NH4HCO3
and incubate 15 minutes. (Occasionally sonicate for 2 to 3
minutes. Add some ice to the sonication water to prevent over
heatting.) |
| c |
Add 20-30 mL acetonitrile to make a 1:1 solution of NH4HCO3/acetonitrile
and incubate for 15 minutes. Remove supernatant and combine wtih
the solution saved in step 5a. |
| d |
To the gel add 20-30 mL of 5% formic acid,
then incubate for 15 minutes. |
| e |
Add the same amount of acetonitrile, then incubate for
15 minutes. |
| f |
Remove supernatant, then combine with
solution from step 5a. |
| g |
Repeat steps d) to f) once. Total extraction
is about 140-210 mL.
|
| h |
To the pooled solution from 5a add 100 mM DTT
to give a final concentration of 1 mM DTT. About 1.4-2.1 mL |
| i |
Completely dry the solution in a speed
vacuum. It should take 4-5 hours at medium temperature. |
| j |
Resuspend the sample in 5-10 mL of 0.1% TFA,
vortex and then incubate at least 10 minutes proceed to the mass
spectrometric analysis. |
Silver Stain In-Gel Digestion
|
1 |
Excision band follow the procedure oulined in
coomassie staining |
|
2 |
Decolorization/Destaining (only for silver
stained gels) |
| |
a Prepare the
following two stock solutions just prior to use:
- 2mL of 100 mM sodium thiosulfate: 49.6 mg in 2mL HPLC water.
- 2mL of 30 mM potassium ferricyanide: 19.75 mg in 2mL HPLC
water.
b Combine solution
a and b in a 1:1 ratio to make 4ml working solution labeled as
"reducing solution".
c Add 200 mL reducing solution to
gel tube, destain until brownish color has disappeared. (2 to 3
minutes).
d Quickly discard the reducing
solution from the tube, add 500 mL of HPLC water to wash. Repeat
wash 2 times, with 5min each.
e Dry samples in speed
vacuum for approximately 15 minutes (until completely dry). |
| 3 |
Reduction and alkylation
Follow procedure as outlined for Coomassie stained gel. |
| 4 |
Digestion same as
Coomassie stain.
Follow procedure as outlined for Coomassie stained gel. |
TOP |
