Staining of Gels for Mass Spectrometric Analysis
EMBL Silver staining
Blum Silver staining
Vorum Silver staining
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Coomassie Brilliant Blue staining

Buffers
Fixer: 45.4% MeOH, 4.6% HAc      vol. 1000 ml.
454 ml MeOH
454 ml H2O
92 ml HAc
Stain: 45.4% MeOH, 4.6% HAc, 0.1% Coomassie     vol. 1000 ml.
454 ml MeOH
454 ml H2O
92 ml HAc
1 g Coomassie Brilliant Blue R-250
Filter solution before use!
Destain: 5% MeOH, 7.5% HAc   vol. 1000 ml.
50 ml MeOH
875 ml H2O
75 ml HAc

 

Procedure

1. Fix gel   100 ml 45.4% MeOH, 4.6% HAc for 1 hour. Do not fix gels for preparative use
2. Stain gel   100 ml 45.4% MeOH, 4.6% HAc, 0.1% Coomassie for 1 hour.
3. Destain gel     100 ml 5% MeOH, 7.5% HAc for 24 hours. Replace if needed.

Zn-imidazole reverse staining

Buffers
Stain: 0.2 M imidazole, 0.1% SDS, pH 8.0   vol. 200 ml.
Stain: 0.2 M Zn-sulphate or 0.2 M ZnCl (pH 2.5)       vol. 200 ml.
Wash: MilliQ water   vol. 200 ml.

 

Procedure
1. Wash gel   Water for 30-60 sec.
2. Soak gel   0.2 M imidazole, 0.1% SDS for 15 mins.
3. Stain gel   0.2 M Zn-sulphate or 0.2 M ZnCl for approx. 30-60 secs.until the gel background becomes white leaving transparent protein bands.
4. Stop staining   3 washes with water.
5. Mobilize protein     0.5 ml 50 mM NH4HCO3, 100 mM DTT for 10-20 min, until the gel plug is completely transparent.

 

EMBL Silver staining

Buffers
Fixer: 50% MeOH, 5% HAc      vol. 200 ml.
100 ml MeOH Merck 1.06009
10 ml HAc (100%) Merck 1.00063
90 ml H2O MilliQ
Wash: 50% MeOH  vol. 200 ml.
100 ml MeOH
100 ml H2O
Sensitizing: 0.02% Na2S2O3 vol. 200 ml.
0.04 g. Na2S2O3 Fluka 72049
200 ml H2O
Silver nitrate: 0.1% AgNO3 Vol. 200 ml. (Cold)
0.2 g AgNO3 Sigma S-8157
200 ml. H2O
Developer: 0.04% Formalin, 2% Na2CO3      Vol. 250 ml.
100 µl Formalin (35% Formaldehyde)      Merck 1.04001
5 g Na2CO3 Merck 1.06392
250 ml. H2O
5% HAc: 5% HAc Vol. 200 ml.
10 ml HAc (100%)
190 ml H2O

 

Procedure
1. Fix gel:   50% MeOH, 5% HAc for 20 mins.
2. Wash gel:   50% MeOH for 10 mins.
3. Wash gel:   H2O for 2 hrs.

Reduce background staining by over night washing.

4. Sensitize gel:   0.02% Na2S2O3 for 1 min.
5. Wash gel:   H2O for 1 min.
6. Wash gel:   H2O for 1 min.
7. Incubate gel:   Cold 0.1% AgNO3 for 20 mins. at 4oC
8. Wash gel:   H2O for 1 min.
9. Change gel chamber
10. Wash the gel:   H2O for 1 min.
11. Develop gel:   0.04% Formalin, 2% Na2CO3

Observe the color and change solution when the developer turns yellow. Terminate when the staining is sufficient.

12. Termination:   5% Hac

Change the solution a couple of times

13. Leave the gel at 4oC in:   1% HAc

  

Blum Silver staining (modified for MS)

Buffers
Fixer: 40% EtOH, 10% HAc, 50% H2O vol. 200 ml.
80 ml EtOH (99.8%)
20 ml HAc (100%)
100 ml H2O (milli-Q)
Wash: 30% EtOH, 70% H2O vol. 200 ml.
60 ml EtOH
140 ml H2O
Sensitizing: 0.02% Na2S2O3 vol. 200 ml.
0.04 g Na2S2O3
200 ml H2O
Silver nitrate: 0.2% AgNO3, 0.02% formalin (cold)     vol. 200 ml.
0.4 g AgNO3
40 µl formalin (35% Formaldehyde)
200 ml H2O
Developer: 3% Na2CO3, 0.05% formalin vol. 250 ml.
7.5 g Na2CO3
125 µl formalin (35% Formaldehyde)
250 ml H2O
5% HAc: 5% Hac vol. 200 ml.
10 ml HAc
190 ml H2O

 

Procedure
1. Fix gel:   40% EtOH, 10% HAc for 1 hr.
2. Wash gel:   30% EtOH, 2 x 20 mins.
3. Wash gel:   H2O for 20 mins.
4. Sensitize gel:   0.02% Na2S2O3 for 1 min.
5. Wash gel:   H2O, 3 x 20 secs.
6. Incubate gel:   Cold 0.1% AgNO3, 20 mins. at 4oC
7. Wash gel:   H2O, 3 x 20 secs.
8. Change gel chamber
10. Wash the gel:   H2O for 1 min.
11. Develop gel:   3% Na2CO3, 0.05% formalin

Observe the color and change solution when the developer turns yellow. Terminate when the staining is sufficient.

12. Wash the gel: H2O for 20 secs.
13. Termination:   5% Hac
14. Wash the gel: H2O, 3 x 10 mins.
15. Leave the gel at 4oC in:   1% HAc

   

Vorum Silver staining (modified for MS)

Reference not available

Buffers
Fixer: 50% MeOH, 12% HAc, 0.05% formalin (35% Formaldehyde)      200 ml
100 ml MeOH (99.8%)
24 ml HAc (100%)
100 µl formalin (35% Formaldehyd)
76 ml H2O (Milli-Q)
Wash: 35% EtOH (96%) 200 ml
73 ml EtOH
127 ml H2O
Sensitizing: 0.02% Na2S2O3 200 ml
0.04 g Na2S2O3
200 ml H2O
Silver nitrate: 0.2% AgNO3, 0.076% formalin (35% Formaldehyde)      200 ml
0.4 g AgNO3
152 µl formalin (35% Formaldehyde)
200 ml H2O
Developer: 6% Na2CO3, 0.05% formalin (35% Formaldehyde), 0.0004% Na2S2O3      400 ml
24 g Na2CO3
200 µl formalin (35% Formaldehyde)
8 ml 0.02% Na2S2O3
392 ml H2O
Stop solution: 50% MeOH, 12% HAc      200 ml
100 ml MeOH
24 ml HAc
76 ml H2O

 

Procedure

1. Fix gel: 50% MeOH, 12% Hac, 0.05% formalin, 2 hrs or overnight
2. Wash gel: 35% EtOH for 20 mins.
3. Wash gel: 35% EtOH for 20 mins.
4. Wash gel: 35% EtOH for 20 mins.
5. Sensitize gel:  0.02% Na2S2O3 for 2 mins.
6. Wash gel: H2O for 5 mins.
7. Wash gel: H2O for 5 mins.
8. Wash gel: H2O for 5 mins.
9. Stain gel: 0.2% AgNO3, 0.076% formalin for 20 mins.
10. Wash gel: H2O for 1 min.
11. Wash gel: H2O for 1 min.
12. Develop gel: 6% Na2CO3, 0.05% formalin, 0.0004% Na2S2O3
13. Stop staining: 50% MeOH, 12% HAc for 5 mins.
14. Leave the gel at 4oC in: 1% HAc