This product has been discontinued as of June 15, 2011 and is no longer available. We recommend an alternative product from Thermo Scientific Pierce Protein Research Product line: 10X Tris-Glycine SDS Buffer or BupH Tris-Glycine-SDS Buffer Packs
I. Reagents 30% acrylamide/bisacrylamide (37.5:1) aqueous solution (stored in the dark) 1.5 M Tris-HCl buffer (pH 8.8) 0.5 M Tris-HCl buffer (pH 6.8) 10% ammonium persulfate (APS) solution (always should be prepared freshly) TEMED 1X Tris-glycine-SDS Buffer (10X buffer diluted to 1X concentration prior use) Caution. Acrylamide is a neurotoxin. Always wear gloves, safety glasses, and a surgical mask when working with acrylamide powder. II. Resolving Gel Preparation
Components
Volume: 10 ml resolving gel solution (for 2 minigels)
for 8% gel
for 10% gel
for 12% gel
Deionized water
4.73 ml
4.13 ml
3.43 ml
30% acrylamide/bisacrylamide
2.7 ml
3.3 ml
4.0 ml
1.5 M Tris-HCl containing 0.4% SDS, pH 8.8
2.5 ml
2.5 ml
2.5 ml
10% APS
60 µl
60 µl
60 µl
TEMED
13 µl
13 µl
13 µl
III. Stacking Gel Preparation
Components
Volume: 5 ml stacking gel solution (for 2 minigels)
Deionized water
3.0 ml
30% acrylamide/bisacrylamide
700 µl
0.5 M Tris-HCl containing 0.4% SDS, pH 6.8
1.25 ml
10% APS
25 µl
TEMED
20 µl
IV. Procedure
Assemble the glass plate sandwich. Prepare the resolving gel solution as described above.
Add APS and TEMED last, mix carefully to avoid formation of bubbles.
Important Note. Polymerization begins as soon as APS is added to the mixture, so all subsequent actions must be performed promptly.
Pour the gel solution between the glass plates with a pipette, leave about 1/4 of the space free for the stacking gel. Carefully cover the top of the resolving gel with 50% isopropanol, 0.1% SDS solution or water, and wait until the resolving gel polymerizes (~30 min). A clear line will appear between the gel surface and the solution on top when polymerization is complete.
Discard the water, isopropanol or SDS solution. Wash gently with double-distilled water.
Pour the stacking gel solution (prepared as described above, add APS and TEMED last) carefully with a pipette to avoid formation of bubbles. Important Note. Polymerization begins as soon as APS is added to the mixture, so all subsequent actions must be performed promptly.
Insert combs. Allow the gel to polymerize for at least 60 min.
Remove combs carefully. Put the gel into the electrophoresis tank, fill the tank (bottom and top reservoirs) with fresh 1X Tris-glycine-SDS Buffer, make sure that the gel wells are covered with the buffer.
Load protein ladder/marker and probes.
Set an appropriate voltage and current depending on how many gels you run. Increase the power when the dye front reaches the running gel. For exact values refer to the table below:
Gel
1 minigel
2 minigels
Stacking gel (upper)
13 mA
25 mA
Resolving gel (lower)
25 mA
50 mA
Values presented are for 0.75 mm gels. For thicker gels the current should be appropriately increased.
Stop the electrophoresis run when the dye front reaches the bottom of the gel. Disassemble the gel sandwich and proceed with gel staining or Western blot procedures.
Semi-dry Protein Transfer for Western Blotting
Note
Wear gloves throughout the procedure to avoid contamination. Use 100 ml of each solution for mini gels (8x10 cm; 10x10 cm), for larger gels use enough of the solution to completely cover the gel/membrane/paper sheets in each step. Buffers Tris-glycine-methanol protein transfer buffer. Cool at 4°C before use.
Component
Amount
Final concentration
10X Tris-glycine buffer
10 ml
1X
Methanol
10 ml
10% (v/v)
Deionized water
to 100 ml
CAPS buffer for electrotransfer of proteins onto PVDF for N-terminal sequencing. Cool at 4°C before use.
Component
Amount
Final concentration
10X CAPS (100 mM, pH 11.0)
10 ml
10 mM
Methanol
10 ml
10% (v/v)
Deionized water
to 100 ml
10X CAPS (3-[cyclohexylamino]-1-propanesulfonic acid). Store at 4°C.
Component
Amount
Final concentration
CAPS
2.21 g
100 mM
Deionized water
to 90 ml
2N NaOH
titrate to pH 11.0 (~4 ml)
Deionized water
to 100 ml
Ponceau S staining solution (only freshly made staining solution should be used).
Component
Amount
Final concentration
Ponceau S
0.2 g
0.2% (w/v)
Glacial acetic acid
1 ml
1% (v/v)
Deionized water
to 100 ml
Semi-dry Protein Transfer
Presoak 2-4 pieces of blotting paper (cut to the size of the gel) in transfer buffer for 5 min.
Cut a piece of nitrocellulose membrane to the size of the gel and equilibrate it in transfer buffer. If a PVDF membrane is used, incubate it in methanol for 2 min before equilibrating it in transfer buffer. Use CAPS buffer for N-terminal sequencing, Tris-glycine-methanol protein transfer buffer is suitable for all other applications.
Carefully remove the stacking gel from the resolving gel. Soak the resolving gel in CAPS buffer for 5 min if this buffer is used. This step can be omitted if Tris-glycine-methanol protein transfer buffer is used.
Assemble the transfer sandwich with the resolving gel on the anode (+) as shown in Figure below. Use one sheet of blotting paper or two pieces of filter paper on each side of the sandwich. Make sure all air bubbles are removed since they will affect the efficiency of the electroblotting.
Electrotransfer proteins from the gel on the membrane for ~60 min at room temperature. Maintain the current at 0.8 mA per 1 cm2 of the gel area and limit the voltage to 15 V.
When the transfer is complete, turn off the power and peel off the layers of the sandwich until you reach the membrane. Remove the membrane with forceps and rinse it in deionized water.
Monitoring the Protein Transfer The efficiency of electrotransfer can be monitored using prestained protein ladders (Spectra™ Multicolor Broad Range Protein Ladder, PageRuler™ Prestained Protein Ladder, PageRuler™ Plus Prestained Protein Ladder and Prestained Protein Molecular Weight Marker. The use of the DualColor™ Protein Loading Pack also allows for monitoring of Western blot protein transfer from gel to membrane. Alternatively, the extent of protein transfer can be determined by staining the polyacrylamide gel after the transfer or by staining the protein directly on the membrane. Proteins on PVDF membranes can be visualized with the PageBlue™ Protein Staining Solution, while Ponceau S, India Ink or Amido Black are recommended for nitrocellulose and PVDF membranes. Figure. Blotting with a semi-dry transfer unit.
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