| 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 ddH?O 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 ddH?O 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 ddH?O to 1000ml |
| Wet transfer buffer | ? |
| 25 mM Tris-base | 3.03 g |
| 192 mM Glycine | 14.4 g |
| 20% Methanol | 200 ml |
| Add ddH?O 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 ddH?O 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% |
| ddH?O | ?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% |
| ddH?O | 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 ddH?O to 1000 ml. | |
| Tricine Gel Recipe | ? | ? | ? |
| Reagents | Stacking | Intermediate | Separating |
| Gel percentage | 4% | 10% | 15% |
| Gel volume | 2ml | 3ml | 6ml |
| 38% Glycerol | - | - | 1.6 |
| ddH?O | 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+2H?O
|
2.9 g | 20 mM Tris-base | 2.4 g |
|
1.9 mM Citric?acid+H?O
|
0.4 g | 150 mM NaCl | 8.7 g |
| Adjust pH to 6.0 | ? | Adjust pH to 7.6 | ? |
| Add ddH?O?to 1000 ml | ? | Add ddH?O?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?C??H??N?Na?O??+2H?O
|
?0.372 g | ?Tween-20 | ?1 ml |
| Adjust pH to 9.0 | ? | ? | ? |
|
Add ddH?O?to 1000 ml
|
?
| 1X PBS | In 1000 ml (final volume) |
| 10 mM Na?HPO? | 1.42 g |
| 1.8 mM NaH?PO? | 0.22 g |
| 140 mM NaCl | 8.18g |
| 2.68mM KCL | 0.20 g |
| Adjust to pH 7.4 | ? |
| Add ddH?O 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 ddH?O 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% ?-mercaptoethanol | 5.0 ml |
| Add ddH?O 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 Na?HPO? | 1.42 g |
| 1.7 mM NaH?PO? | 0.20 g |
| 140 mM NaCl | 8.19 g |
| Add ddH?O 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 ddH?O to 1000 ml | |
| PBST buffer |
For 1000 ml
|
| 58 mM Na?HPO? | 8.24 g |
| 17 mM NaH?PO? | 2.04 g |
| 68 mM NaCl | 3.98 g |
| 1%Triton-X100 | 10 ml |
| Adjust to pH 7.4 | |
| Add ddH?O 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 ddH?O to 1000 ml | |
| PBS with 2% Sarkosyl | For 200 ml |
| 58 mM Na?HPO? | 1.65 g |
| 17 mM Na?HPO? | 0.41 g |
| 68 mM NaCl | 0.80 g |
| 2% Sarkosyl | 4 .00 g |
| Adjust to pH 8.0 | |
| Add ddH?O to 200 ml | |
| 8 M Urea | For 200 ml |
| Urea | 96.08 g |
| Adjust to pH 8.0 | |
| Add ddH?O to 200 ml | |