Tris-glycine-methanol protein transfer buffer
Reagent
Amount Final concentration 10X Tris-glycine buffer (#B47) 0.3g 25 mM Glycine 1.44g 192 mM Methanol 10 ml 10% (v/v) deionized water to 100 ml Cool at 4°C before use.
CAPS buffer for electrotransfer of proteins onto PVDF for N-terminal sequencing
Reagent
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 Cool at 4°C before use.
10X CAPS
Reagent
Amount Final concentration CAPS (3-[cyclohexylamino]-1-
propanesulfonic acid)4.43g 100 mM deionized water to 90 ml 2N NaOH titrate to pH 11.0 (~4 ml) deionized water to 100 ml Store at 4°C.
Note Wear gloves throughout the procedure to avoid contamination.
Use 100 ml of each solution for minigels, while for larger gels use enough of the solution to cover completely the gel/membrane/paper sheets in each soaking step.
1. Presoak two-four pieces of blotting paper (cut to the size of the gel) in the transfer buffer. 5 min 2. Cut a piece of nitrocellulose membrane to the size of the gel and equilibrate it in the transfer buffer. If a PVDF membrane is used, rinse it in methanol for two minutes before equilibrating it in the transfer buffer. Use the CAPS buffer for the N-terminal sequencing, while the Tris-glycine buffer is suitable for all other applications. 5 min 3. Carefully remove the stacking gel from the resolving gel. Soak the resolving gel if the CAPS buffer is used. This step can be omitted with the Tris-glycine buffer. 5 min 4. Assemble the transfer sandwich with the resolving gel on the anode (+) as shown in picture below. Use one sheet of blotting paper or two pieces of filter paper on each side of the sandwich. Make sure you remove all air bubbles since they will affect the efficiency of the electroblotting. 5. Electrotransfer proteins from the gel on the membrane at room temperature. Keep the current at 0.8 mA per 1 cm2 of gel and limit the voltage to 15V. 60 min 6. When the transfer is complete, turn off the power and peel off the layers of the sandwich until you reach the membrane. 7. Remove the membrane with a pair of forceps, rinse in deionized water. 1 min
Blotting with a semi-dry transfer unit.Monitoring the Protein TransferThe efficiency of electrotransfer can be monitored using prestained protein markers (see PageRuler™ Prestained Protein Ladder). 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 nitrocellulose and PVDF membranes can be stained with India Ink, Amido Black or Ponceau S and other dyes.
Staining of PVDF Membrane
- Staining using PageBlue™ Protein Staining Solution
Note PVDF membrane must be dry.
1. Add PageBlue™ Protein Staining Solution (#R0571) to cover the PVDF membrane. Agitate gently. 2 min 2. Wash with 30% ethanol with gentle agitation. 5 min 3. To remove the stain completely, wash the membrane with the mixture of 30% acetonitrile and 20% ethanol. 5 min
- Ponceau S Staining
Ponceau S staining solution
Reagent
Amount Final concentration Ponceau S 0.2g 0.2% (w/v) Glacial acetic acid 1 ml 1% (v/v) deionized water to 100 ml
1. If the membrane is dry, rehydrate it with water for 5 minutes (or methanol if using PVDF). 5 min 2. Stain the membrane with the Ponceau S staining solution under agitation. 5 min 3. Destain the membrane under agitation with deionized water until bands are visible. ~5 min 4. If required, wash the blot with 0.1N NaOH to remove the stain completely. ~5 min
Ordering Information PageRuler™ Prestained Protein Ladder #SM0671 2x250 µl PageBlue™ Protein Staining Solution #R0571 1 liter (for up to 150 mini-gels)
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Updated gegužės 10, 2007 11:24
