Detailed protocol

A. General Comments
1. To reduce the possibility of amino-terminal blockage of the protein:

a. use the highest purity gel reagents possible
b. make fresh stock solutions for important samples
c. age gel prior to running protein
d. reducing agents should be present during electrophoresis

2. Wear gloves. Handle gels and blots only at the edges to minimize background contamination.3. The concentration of acrylamide in the lower gel (see B. below) can be from 7-18% but 12.5% is the most useful for 10-70Kd proteins.4. Use a mini-gel system to maximize protein-to-gel ratio and load as concentrated a protein solution as possible without losing resolution. One wants to load the sequencer with as much protein and as little membrane as possible for best results.

5. Run a set of MW markers on each gel to serve as controls in case the unknown protein is blocked. One can sequence the first few residues of a marker band (ie. lysozyme at 14.3 kD) to make sure the gel reagents didn’t chemically block the protein.

6. If identification of “cys” residues is desired, consider reduction
and alkylation of the sample in the gel loading buffer.

B. Tris-Glycine Gels
1. Pour lower gel, cover with H2O sat’d butanol to break any bubbles and let sit overnight.2. Pour off butanol, flush with water several times and dry completely with wipe.3. Pour upper gel, let sit 30 min then flush wells by filling with water and shaking sharply; repeat two more times.

4. Dissolve protein in reducing sample loading buffer (r-S.L.B.) at 1-2 µg / 10 ul and let reduce for 10 min at 60 C.

5. Load the entire sample and run at 25 mA (constant current) for about 1 hour until the dye marker is near the bottom of gel.

6. If the sample is very dilute, pour an extra long stacker. Completely fill the wells with the sample and run until the dye has just entered the stacker. Flush the wells with running buffer and load another aliquot. If needed, a third aliquot can usually be loaded without compromising resolution.

Recipe for 12.5 % gels:

Lower gel Upper gel
30% acrylamide/0.8% bis
4.20 ml
0.75 ml
4X U.G.B.
1.25 ml
4X L.G.B.
2.50 ml
water
3.10 ml
2.90 ml
10% NH4 persulfate (fresh)
0.20 ml
0.10 ml
TEMED
10 µl
5 µl
TOTAL
10.00 ml
5.00 ml
C. Electroblotting onto a PVDF Membrane
1. Carefully remove the upper gel from the lower gel and briefly place the lower gel in a tray of blotting buffer.2. Wet precut pieces of membrane with 100% methanol for a few seconds then transfer to blotting buffer in a second tray. Make sure the membrane is completely wetted.3. Dip the sponges and precut filter paper pieces in blotting buffer in a third tray.

4. Assemble the blotting sandwich as follows:
sponge // filter paper // gel // PVDF // filter paper // sponge

a. make sure there are no trapped air bubbles between any of the layers
b. assemble the sandwich on its holder so that the gel is toward the neg. (usually black) electrode and the membrane is toward the positive (usually red) electrode.
c. make sure the sponges are in good condition; if the blotting cassette is easy to close, the sponges are no good.

5. Fill the blotting apparatus with blotting buffer and apply a constant voltage (ca. 70V) for 30 to 40 min.

a. one can also transfer overnight at a low constant current (ca. 15 mA) in a cold room.

D. Staining the PVDF Membrane
1. Remove the membrane from the sandwich, briefly rinse with water then soak it in 100% methanol for a few seconds.2. Stain the membrane in dye (Coomassie) solution with constant shaking for no more than 60 sec.

a. Ponceau red and amino black are acceptable alternatives toCoomassie but are generally less sensitive.

3. Immediately destain in 50% methanol with constant shaking , changing the solution every couple of minutes until the solution remains colorless.4. Rinse the membrane thoroughly with water and let dry 30-60 min. to increase the contrast of the bands. 
E. Stock Solutions
4X U.G.B. 6.05 gm Tris pH=6.8 (ca. 7 ml 6N HCl)
0.40 gm SDS
to 100. ml with water
4X L.G.B. 18.17 gm Tris pH=8.8 (ca. 4 ml 6N HCl)
0.40 gm SDS
to 100. ml with water
2X S.L.B. 12.5 ml 4X U.G.B.
20.0 ml 50% glycerol
3.0 gm SDS
to 45. ml with water
Reducing S.L.B.: 0.45 ml 2X S.L.B.
0.45 ml water
0.05 ml 2-mercaptoethanol
0.05 ml 0.1% BPB dye solution
Tank buffer: 30.3 gm Tris pH=8.0
(10X stock) 144.1 gm glycine
10.0 gm SDS
to 1000. ml with water
Blotting Buffer: 100. ml 10X CAPS buffer
100. ml methanol
800. ml water
10X CAPS: 22.13 gm CAPS pH=11 (2N NaOH)
(store at 4 C.) to 1000. ml with water
Dye Solution: 1. dissolve 1.0 gm Commassie blue in 400 ml methanol
and stir for 1 hour.
2. add 10 ml acetic acid and 590 ml water.
3. stir for 30 min and filter thru a 0.45 µm filter.

Please click here if this helped you.
10 people found this helpful.


Category: Edman Sequencing

← Detailed protocol
Posted in