Running
Buffer Recipes.
10X Tris-glycine-SDS buffer (denaturing conditions; Laemmli)
Ingredient |
Amount |
Final Stock Concn |
Tris-base |
30.2 g |
0.250 M |
Glycine |
144.0 g |
1.92 M |
SDS |
10.0 g |
1% (w/v) |
distilled water |
to 1 liter |
. |
For use, dilute 1 in 10 with distilled water. Working
solution is pH 8.3
10X Tris-glycine buffer
Ingredient |
Amount |
Final
Stock Concn |
Tris-base |
30.2 g |
0.250 M |
Glycine |
144.0 g |
1.92 M |
distilled water |
to 1 liter |
. |
For use, dilute 1 in 10 with distilled water. Working
solution is pH 8.3
5X Tris-tricine-SDS buffer
Note: IDGel™ Precast Gels are suitable for use with the Tris-tricine
buffer system (1). Use of this buffer improves the separation of peptides
and small proteins
Ingredient |
Amount |
Final
Stock Concn |
Tris-base |
60.5 g |
0.5 M |
Tricine |
89.6 g |
0.5 M |
SDS |
5.0 g |
0.5% |
distilled water |
to 1 liter |
. |
For use, dilute 1 in 5 with distilled water. Working
solution is pH 8.25
(1) Shagger, H., and G. von Jagow. Tricine-sodium dodecyl
sulfate-polyacrylamide gel electrophoresis for the separation of proteins
in the range from 1 to 100 kDa.
Anal Biochm 166:368-379, 1987
10X Tris-acetate-EDTA buffer
Ingredient |
Amount |
Final
Stock Concn |
Tris-base |
48.4 g |
0.4 M |
glacial acetic acid |
11.42 ml |
0.2 M |
EDTA 0.5M (pH 8.0) |
20.0 ml |
0.01 M |
distilled water |
to 1 liter |
. |
For use, dilute 1 in 10 with distilled water.
10X Tris-borate-EDTA buffer
Ingredient |
Amount |
Final
Stock Concn |
Tris-base |
108.0 g |
0.112 M |
boric acid |
55.0 g |
0.112 M |
EDTA 0.5M (pH 8.0) |
40.0 ml |
0.02 M |
distilled water |
to 1 liter |
. |
IDGel™ Western procedure.
Western Blotting.
Note: The following protocols for Western blotting provide general
information and conditions that may be applied to most transfer procedures.
However, careful optimization should be considered if quantitative
recovery of a particular protein is required since blotting efficiency
is dependent on buffer composition, blotting time and current and membrane
used.
Transfer with a tank blotting system
Transfer buffer*
Ingredient |
Amount |
Final Stock Concn |
Tris-base |
3 g |
25 mM |
Glycine |
14.4 g |
192 mM |
Methanol** |
100 - 200 ml |
10 - 20 % |
distilled water |
to 1 liter |
. |
*cool to 4°C before use
**10% methanol is recommended when using PVDF blotting membranes
while 20% is recommended when using nitrocellulose-blotting membranes.
Transfer protocol
- Pre-soak the porous pads and two pieces of filter paper cut to
the size of the gel (ex. Whatman 3MM paper) in transfer buffer for
5 min
- Cut a piece of blotting membrane to the size of the gel and equilibrate
in transfer buffer. If a PVDF membrane is used, rinse in methanol
for 2 min before equilibration in the transfer buffer for 5 min
- Remove the gel from the cassette and equilibrate in transfer buffer
for 5 min
- Carefully assemble the transfer stack. Remove all air bubbles between
the layers. If you do NOT do this then the air bubbles will affect
the efficiency of the transfer process and may leave blank spots
on the blotting membrane
- Place the stack into the tank unit making sure it is in the PROPER
ORIENTATION. Fill the chamber with transfer buffer
- Transfer at room temperature for either 60 min at 150 mA
or 90 min at 75 mA (or 100 volts).
- When the transfer is complete, turn off the power and remove the
layers until you reach the blotting membrane
- Remove the blotting membrane with a pair of forceps, rinse in distilled
water and air-dry for 1 min.
Transfer with a semi-dry system
Transfer buffer
Ingredient |
Amount |
Final Stock Concn |
Tris-base |
0.3 g |
25 mM |
Glycine |
1.44 g |
192 mM |
Methanol |
10.0 ml |
10% (v/v) |
distilled water |
to 100 ml |
. |
*cool to 4°C before use
Transfer protocol
(Note: Wear gloves throughout the procedure)
- Pre-soak four pieces of blotting paper and one transfer membrane
to the size of the gel and equilibrate in transfer buffer. If a PVDF
membrane is used, rinse it in methanol for 2 min before equilibration
in the transfer buffer for 5 min
- Remove the gel from the cassette. Carefully assemble the transfer
stack on the anode (+). Remove all air bubbles between the layers.
Air bubbles will affect the efficiency of the transfer process and
may leave blank spots on the blotting membrane
- Transfer at room temperature for either 30 min at 200 mA,
60 min at 100 mA or 90 min at 75 mA (or 100 volts).
- When the transfer is complete turn off the power and remove layers
until you reach the blotting membrane
- Remove the blotting membrane with a pair of forceps, rinse in distilled
water and air-dry for 1 min
Monitoring of protein transfer
The efficiency of transfer can be monitored using prestained protein
markers. Alternatively, the extent of protein transfer can be determined
by staining the polyacrylamide gel after the transfer is complete or
by staining the proteins directly on the blotting membrane. Proteins
on solid support can be stained with dyes such as India ink, Amido
Black or Coomassie Blue. A recommended stain is Ponceau S, which is
reversible. The detection limit is 1 to 2 micrograms.
Ponceau S staining for proteins on solid support
Ingredient |
Amount |
Final Stock Concn |
Ponceau S |
0.2 g |
0.2% (w/v) |
Glacial Acetic Acid |
1 ml |
1% (v/v) |
distilled water |
to 100 ml |
. |
Protocol
- If the blotting membrane is dry, rehydrate it with water (or methanol,
if PVDF) for 5 min
- Stain the blotting membrane under constant shaking with the Ponceau
S solution for 5 min
- Destain the membrane under constant shaking with distilled water
- If required, wash the blot with 0.1N NaOH to remove the stain completely
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