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RIPA lysis buffer | For 1000 ml |
50 mM TrisHCl, pH 7.4 (1 M stock) | 50 ml |
150 mM NaCl | 8.76 g |
1% Triton X-100 | 10 ml |
0.5% Sodium Deoxylcholate | 5 g |
0.1 % SDS | 1 g |
10 mM NaF | 0.41 g |
1 mM EDTA (0.5 M stock) | 2 ml |
Add ddH₂O to 1000 ml | |
Adjust to pH 7.4 | |
Add PMSF to 1 mM and other protease inhibitors immediately prior to use. |
4X SDS sample buffer | |
150 mM Tris•HCl (pH 7.0) (1M stock) | 15 ml |
25% Glycerol | 25 ml |
12% SDS | 12 g |
0.05% Bromophenol Blue | 0.05g |
6% β-mercaptoethanol
|
6 ml |
Add ddH₂O to 100ml, aliquot and store at -20°C |
1X TBST | |
20 mM Tris-base | 2.42 g |
150 mM NaCl | 8.76 g |
50 mM KCl | 3.73 g |
0.2% Tween-20 | 2 ml |
Adjust pH to 7.6 | |
Add ddH₂O to 1000ml |
Wet transfer buffer | |
25 mM Tris-base | 3.03 g |
192 mM Glycine | 14.4 g |
20% Methanol | 200 ml |
Add ddH₂O to 1000ml | |
Semi-dry transfer buffer | |
48 mM Tris-base | 5.81 g |
39 mM Glycine | 2.93 g |
0.0375% SDS | 0.375 g |
20% Methanol | 200 ml |
Add ddH₂O to 1000ml |
Separating gel (ml, total 10 ml) | ||||
MW of target protein (kDa)
|
80-200 | 35-100 | 25-60 | 20-40 |
Gel percentage | 8% | 10% | 12% | 15% |
ddH₂O | 2.1 | 1.5 | 0.8 | 0 |
30% Acrylamide
|
2.7 | 3.3 | 4 | 5 |
2x Separating buffer
|
5.0 | 5.0 | 5.0 | 5.0 |
10% APS | 0.1 | 0.1 | 0.1 | 0.1 |
TEMED | 0.01 | 0.01 | 0.01 | 0.01 |
Stacking gel (ml) | 4 ml | 6ml | 8ml |
MW of target protein (kDa)
|
- | - | - |
Gel percentage | 4% | 4% | 4% |
ddH₂O | 1.4 | 2.1 | 2.7 |
30% Acrylamide | 0.5 | 0.8 | 1.1 |
2x Stacking buffer | 2.0 | 3.0 | 4.0 |
10% APS | 0.04 | 0.06 | 0.08 |
TEMED | 0.004 | 0.006 | 0.008 |
2x Separating Buffer Recipe (makes 1000ml) | |
Tris HCl (pH 8.8) | 90.8 g |
SDS | 2.0 g |
Dissolve compounds thoroughly. Adjust pH slowly to pH 8.8 with concentrated HCl, then add ddH2O to 1000ml. |
2x Stacking Buffer Recipe (makes 1000ml) | |
Tris HCl (pH 6.8) | 30.35 g |
SDS | 2.0 g |
Dissolve compounds thoroughly. Adjust pH slowly to pH 6.8 with concentrated HCl, then add ddH2O to 1000ml.
|
1x Running Buffer Recipe (makes 1000ml) | |
Tris-base | 1.51 g |
Glycine | 7.5 g |
SDS | 0.5 g |
Dissolve compounds thoroughly, then add ddH₂O to 1000 ml. |
Tricine Gel Recipe | |||
Reagents | Stacking | Intermediate | Separating |
Gel percentage | 4% | 10% | 15% |
Gel volume | 2ml | 3ml | 6ml |
38% Glycerol | - | - | 1.6 |
ddH₂O | 1.4 | 1.2 | - |
30% Acrylamide | 0.3 | 0.8 | 2.7 |
3.0 M Tris HCl (pH 8.5)
|
- | 1 | 2.14 |
1.0 M Tris HCl (pH 6.8)
|
0.3 | - | - |
10% SDS | 0.02 | 0.03 | 0.06 |
10% APS | 0.02 | 0.03 | 0.06 |
TEMED | 0.002 | 0.003 | 0.003 |
Citrate buffer | For 1000 ml | 1x TBS | For 1000 ml |
10 mM Trisodium citrate+2H₂O
|
2.9 g | 20 mM Tris-base | 2.4 g |
1.9 mM Citric acid+H₂O
|
0.4 g | 150 mM NaCl | 8.7 g |
Adjust pH to 6.0 | Adjust pH to 7.6 | ||
Add ddH₂O to 1000 ml | Add ddH₂O to 1000 ml |
Tris-EDTA (TE) buffer | For 1000 ml | 1x TBST | For 1000 ml |
10 mM Tris-base | 1.21 g | 1x TBS | 999 ml |
1 mM EDTA C₁₀H₁₄N₂Na₂O₈ +2H₂O
|
0.372 g | Tween-20 | 1 ml |
Adjust pH to 9.0 | |||
Add ddH₂O to 1000 ml
|
1X PBS | In 1000 ml (final volume) |
10 mM Na₂HPO₄ | 1.42 g |
1.8 mM NaH₂PO₄ | 0.22 g |
140 mM NaCl | 8.18g |
2.68mM KCL | 0.20 g |
Adjust to pH 7.4 | |
Add ddH₂O to 1000 ml |
Antibody dilution buffer | In 20 ml (final volume) |
1% BSA | 0.2g |
Add 1X PBS to 20ml | |
RIPA lysis buffer | For 1000 ml |
50 mM TrisHCl, pH 7.4 (1 M stock) | 50 ml |
150 mM NaCl | 8.76 g |
1% Triton X-100 | 10 ml |
0.5% Sodium Deoxylcholate | 5 g |
0.1 % SDS | 1 g |
10 mM NaF | 0.41 g |
1 mM EDTA (0.5 M stock) | 2 ml |
Add ddH₂O to 1000 ml | |
Adjust to pH 7.4 | |
Add PMSF to 1 mM and other protease inhibitors immediately prior to use. |
5X SDS sample buffer | |
250 mM Tris HCl (pH 7.0) (1M stock) | 12.5 ml |
35% Glycerol | 17.5 m |
10% SDS | 5 g |
0.02% Bromophenol Blue | 10 mg |
10% ß-mercaptoethanol | 5.0 ml |
Add ddH₂O to 50ml, aliquot and store at -20°C |
Blocking Buffer | 1000 ml |
Bovine serum albumin | 5.00 g |
1x PBS buffer | 1000 ml |
PBS Buffer | 1000 ml |
10 mM Na₂HPO₄ | 1.42 g |
1.7 mM NaH₂PO₄ | 0.20 g |
140 mM NaCl | 8.19 g |
Add ddH₂O to 1000 ml | |
Adjust to pH 7.4 |
TNMFX-2M Urea | For 1000 ml |
50 mM Tris-base | 6.06 g |
150 mM NaCl | 8.77 g |
1 mM EDTA | 0.37 g |
2 M Urea | 120.20 g |
Adjust to pH 8.0 | |
Add ddH₂O to 1000 ml |
PBST buffer |
For 1000 ml
|
58 mM Na₂HPO₄ | 8.24 g |
17 mM NaH₂PO₄ | 2.04 g |
68 mM NaCl | 3.98 g |
1%Triton-X100 | 10 ml |
Adjust to pH 7.4 | |
Add ddH₂O to 1000 ml |
TNMFX-0.1% Triton X100 | For 1000 ml |
50 mM Tris | 6.06 g |
150 mM NaCl | 8.8 g |
1 mM EDTA | 0.4 g |
0.1% Triton-X100 | 1 ml |
Adjust to pH 8.0 | |
Add ddH₂O to 1000 ml |
PBS with 2% Sarkosyl | For 200 ml |
58 mM Na₂HPO₄ | 1.65 g |
17 mM Na₂HPO₄ | 0.41 g |
68 mM NaCl | 0.80 g |
2% Sarkosyl | 4 .00 g |
Adjust to pH 8.0 | |
Add ddH₂O to 200 ml |
8 M Urea | For 200 ml |
Urea | 96.08 g |
Adjust to pH 8.0 | |
Add ddH₂O to 200 ml |