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1 mini gel
| % | 7.0 | 10 | 12 | 15 | 20 |
| LGB | 1.34 ml | 1.34 ml | 1.34 ml | 1.34 ml | 1.34 ml |
| DDW | 2.5 ml | 2.0 ml | 1.65 ml | 1.0 ml | 0.35 ml |
| 30% | 1.16 ml | 1.66 ml | 2.0 ml | 2.5 ml | 3.35 ml |
| APS 25% | 10 ul | 10 ul | 10 ul | 10 ul | 10 ul |
| TEMED | 10 ul | 10 ul | 10 ul | 10 ul | 10 ul |
2 mini gels
| % | 7.0 | 10 | 12 | 15 | 20 |
| LGB | 2.67 ml | 2.67 ml | 2.67 ml | 2.67 ml | 2.67 ml |
| DDW | 5.00 ml | 4.00 ml | 3.33 ml | 2.0 ml | 0.7 ml |
| 30% | 2.33 ml | 3.33 ml | 4.0 ml | 5.0 ml | 6.7 ml |
| APS 25% | 20 ul | 20 ul | 20 ul | 20 ul | 20 ul |
| TEMED | 20 ul | 20 ul | 20 ul | 20 ul | 20 ul |
3 mini gels
| % | 7.0 | 10 | 12 | 15 | 20 |
| LGB | 4.0 ml | 4.0 ml | 4.0 ml | 4.0 ml | 4.0 ml |
| DDW | 7.5 ml | 6.0 ml | 5.0 ml | 3.0 ml | 1.0 ml |
| 30% | 3.5 ml | 5.0 ml | 6.0 ml | 8.0 ml | 10.0 ml |
| APS 25% | 30 ul | 30 ul | 30 ul | 30 ul | 30 ul |
| TEMED | 30 ul | 30 ul | 30 ul | 30 ul | 30 ul |
4 mini gels
| % | 7.0 | 10 | 12 | 15 | 20 |
| LGB | 5.4 ml | 5.4 ml | 5.4 ml | 5.4 ml | 5.4 ml |
| DDW | 10.00 ml | 8.00 ml | 6.66 ml | 4.0 ml | 1.34 ml |
| 30% | 4.66 ml | 6.66 ml | 8.0 ml | 10.0 ml | 13.7 ml |
| APS 25% | 20 ul | 20 ul | 20 ul | 20 ul | 20 ul |
| TEMED | 20 ul | 20 ul | 20 ul | 20 ul | 20 ul |
Stacking gels
| 1 gel | 2 gels | 3 gels | 4 gels | ||
| UGB | 0.61 ml | 1.25 ml | 1.86 ml | 2.5 ml | |
| DDW | 1.5 ml | 3.0 ml | 4.5 ml | 6.0 ml | |
| 30% | 0.4 ml | 0.8 ml | 1.2 ml | 1.6.0 ml | |
| APS 25% | 10 ul | 20 ul | 30 ul | 40 ul | |
| TEMED | 10 ul | 20 ul | 30 ul | 40 ul | |
1. Remove gel from plates
2. Equilibrate gels in 0.5 x transfer buffer ± 10% methanol for 10 min (Avoid use of methanol with large proteins (>100Kd, but include it with smaller proteins).
10 X Western transfer buffer
30.28 g Tris (0.25M)
144.88 g Glycine (1.93M)
Add DDW to 1 Lt
pH should be ~pH 8.3
Do not adjust pH
3. Wet small (8.5cm x 6cm), large (10cm x 7cm) pieces 3MM paper and 1 piece Hybond C-super (9cm x 6cm).
4. Assemble transfer apparatus according to Millipore manual ie 3 large 3MM, filter, equilibrated gel with upper gel removed, 3 small 3MM on the "base" of the transfer apparatus
5. Transfer for duration according to Millipore manual and at mAmp calculated by: 1.2 mA/cm2 x area of gel for 1 hr or 2.5 mA/cm2 x area of gel for 45 min. One small gel = 62.5mA/1hr
Remove filter and block for 30 min with:
TBST for binding with biotinylated proteins
PBS/5% Blotto for antibodies
6. Coomassie stain gel to determine if all the protein has transfered
Antibody western:
1. Block with PBS/5% Blotto for 30' to overnight.
2. Add antisera at appropriate dilution (1:200 - 1:2000) to PBS/5% Blotto and add to the filter in a bag.
3. Incubate at RT on the wheel or shaker for 1hr+ (o/n incubations generally give higher backgrounds).
4. Wash 3 X 10' PBS/5% Blotto
5. Incubate with goat anti - rabbit or mouse (for monoclonals) coupled to HRP at 1:1000 - 1:2000 in PBS/5% BLOTTO. We generally use BioRad GAR #1706515 or GAM #1706516.
7. Wash 3 X 10' 5% PBS/BLOTTO
8. Develop the complex with ECL reagent (Amersham #RPN 2106). Combine about 1.5ml of each of the two components for a standard mini gel filter (about 3ml ea. for a 15 X 15cm filter). Incubate for 1' in a bag, squeeze out the excess, seal and autoradiograph with fluorescent dots as markers.
Probing with a biotinylated protein
1. Block filter 30 min TSTG. TSTG (1 Lt) = 10 ml 1 M Tris pH 8.0, 30 ml 5 M NaCl, 0.5 g Tween 20, 1 g Gelatin. Add ~200 ml DDW, heat to 80 - 90 ¡ to dissolve gelatin and add DDW to 1Lt
2. Probe 1 hr room temperature with 5-20ug biotinylated protein in 10 ml TSTG. 3 x 5 min washes TSTG
3. To create an avadin-biotin-HRP complex add 12.5 ul A Soln (Avidin) and 12.5 ul B Soln (Horse radish peroxidase) of the VectorStain kit Immuno Diagnostics #PK 4001 to 10 ml TSTG and allow the complex to form over 30 min without additional mixing. Probe with Avidin/HRP complex 1 hr room temperature. 3 x 5 min washes TSTG and then ECL as above.
Stripping filters
Stripping buffer:
100mM 2-mercaptoethanol
2% SDS
62.5mM Tris HCl pH 6.7
1. Submerge filter in stripping buffer and incubate at 50¡C for 30min. with agitation.
2. Wash filter 3 X 10min with PBS or TSTG at r/t, then ECL to check for removal of the antibody.
3. Reblock filter with 5% BLOTTO in PBS or TSTG at r/t 30min.
4. Reprobe as above.