Polyacrylamide Gel Staining
Notes
- Before pouring a gel, the acrylamide
solution needs to be filtered.
- All buffers (gel-loading buffer, gel-running buffer) used for
the gel running must be filtered using Millipore 0.22 mm presterilized
filter.
- Totally clean the gel stain container prior to use.
Standard Coomassie Staining
|
1 |
Chemical
reagents
Brilliant Blue R-250 (BBR) (Fisher
BP101-25)HPLC water or Milli-Q water. |
| 2 |
Solutions
Fixing solution: 50:10:40 (methanol: acetic acid: H2O)
Destaining solution: 45:10:45 (methanol: acetic acid: H2O)
Coomassie concentrated stain solution: 12.0 g BBR + 300 mL Methanol dissolved, then add 60 mL acetic acid. Stir well.
Coomassie Working solution: 500 mL Methanol+ 30 mL Coomassie concentrated stain solution + 400 mL milli-Q water + 100 mL acetic acid . Mix up and filter using 0.22 mm pre-sterilized filter.
|
| 3 |
Gel
staining protocol
Fix gel in fixing solution for about 30
min.
Stain gel in Coomassie working solution for about 25-40 minutes.
Destain until no background (shaking about 2-3 hours).
Store gel in 5% acetic acid solution at 4°C until in-gel digestion is performed (Gel can be stored for several weeks).
|
Gel-Code Blue Coomassie Staining
Gel-Code Blue Staining has the same sensitivity as standard Coomassie Staining. This procedure utilizes many of the features of colloidal
Coomassie, but the protocol is compressed into 2 main steps allowing visualization of bands within an hour. This procedure is perfect for recombinant proteins or high abundance samples.
|
1 |
Chemical
reagents
GelCode Blue stain Reagent (PIERCE Cat. 24590 or 24592)
HPLC water or Mill-Q water. |
| 2 |
Gel staining protocol
Fix gel in fixing solution (50:10:40 / methanol:acetic acid:H2O) for 30
min.
Wash the gel with 3 alquots of H2O, shaking for 5
min. each.
Stain the gel in GelCode Blue stain Reagent for 1 hour, gently rock at room temperature.
Wash the gel with ddH2O, shake about 2-3 hours, change water 3 to 4 times.
Store gel in 5% acetic acid solution at 4°C until in-gel digestion is performed(Gel can be stored for several weeks). |
Colloidal Coomassie staining
This staining procedure is used for SDS-PAGE or 2D SDS-PAGE gels in
which low background staining and a high degree of sensitivity is
required. While having a sensitivity threshold close to that of silver
staining one needs to keep in mind that the time required for staining
is substantially greater than that of either standard silver or
Coomassie
staining.
In order to see optimal results from this staining protocol one should
allow 3 to 4 days for completion.
|
1 |
Chemical
reagents
Coomassie Brilliant Blue G250 (Biorad)
HPLC water or Mill-Q water. |
| 2 |
Solutions
Fixing solution: 50:3:47 (ethanol: phosphoric acid:
H2O)
Neuhoffs solution: 16:25:5:54 (Ammonium Sulfate: Methanol:
phosphoric acid: H2O) |
| 3 |
Gel staining protocol
Allow gels to fix in this solution overnight. (as little as 4 hours will do and fixation can be prolonged out to 4 days if need be).
Removal of excess fixative and Wash
gels three times for 30 minutes per wash.
Equilibrate gels in Neuhoffs solution
for one hour.
Add Coomassie Brilliant Blue G250 powder (1 g/L) to each staining tray and stain for 3 days. Normally spots can be seen by 24-48 hours but for optimal staining stain for 3-4 days.
Store gel in 5% acetic acid solution at 4°C until in-gel digestion is performed (Gel can be stored for several weeks).
|
Silver Staining
Adapted from Blum et al. Electrophoresis, 8, 93-99, 1987.
This staining protocol is compatible with mass spectrometric protein
analysis (Note: not all silver staining procedures are compatible
with mass spectrometry). The stain is useful for protein concentrations
ranging from approximately 5 ng to 1 mg. If the staining does not
work, gels can be destained
and restained again (see below).
It should be noted that while this method of staining is more sensitive
and much faster than colloidal Coomassie staining, the recovery
of peptides from an in-gel digest is decreased.
|
1 |
Chemical
reagents
Silver nitrate (Fisher S181-25, or sigma S-8157)
HPLC-water or Milli-Q water.
HPLC-grade methanol
Acetic acid
Sodium Thiosulphate
Sodium Carbonate
37% Formaldehyde
Ethylenediaminetetraacetic
acid, disodium salt (Na2-EDTA)
Potassium Ferricyanide (for destaining)
|
| 2 |
Gel
staining protocol
Prepare all solutions
and reagents just prior to use!
|
Step |
Reagent |
Final volume =100 mL
|
Operation |
Time |
| step A |
50% methanol 10% acetic acid
|
|
fix |
30 min |
| step B |
5% methanol |
incubate |
15 min |
| |
milli-Q H2O |
wash 3 times |
3 x 5 min |
| step C |
sodiumthiosulphate
(Na2S2O3.5H2O)
|
0.2 g/L fresh! cold!
|
Incubate |
120 sec |
| |
milli-Q H2O
|
|
wash 3 times |
3 x 30 sec |
| step D |
silver nitrate (AgNO3)
|
0.2 g/100 ml fresh! cold!
|
Incubate |
25 min |
| |
milli-Q H2O
|
|
wash 3 times |
3 x 60 sec |
| step E |
sodium carbonate (Na2CO3)
37% Formaldehyde Solution
Na2S2O3.5H2O
(sol.C) |
3 g/100 ml
50 ul/100 ml
2 ml/100 ml fresh! cold! |
develop |
10 min (max.) |
| step F |
Na2-EDTA
|
(14g / L) |
stop develop |
10 min |
| |
milli-Q H2O
|
|
wash |
|
|
| 3 |
Destaining
protocol
Dissolve 0.4g potassium ferricyanide (K3Fe(CN)6)
in 200ml sodiumthiosulphate solution(0.2 g/L,
step
c above).
Destain:
- Destain until no bands are visible; the gel may be
slightly yellow.
- Wash gel 4-5 times for 15 min with milli-Q H2O
until gel is transparent and has
no background colour.
- Starting with
step
c from the gel staining protocol, restain gel.
|
| 4 |
Gel
Storage
Gel can be stored in 5% acetic acid solution at 4°C for
several weeks prior to in-gel digestion. Alternatively gel pieces
can be stored dry (see procedure
for in-gel digestion).
|
TOP |
