Buffers & Reagents
"Tris" means "tris(hydroxymethyl)aminomethane" in following tables.
Stock for A buf
| 1 M HEPES-KOH (pH7.9) |
1 mL |
10 mM |
| 1 M MgCl2 |
150 µL |
1.5 mM |
| 3 M KCl |
333 µL |
10 mM |
| DW |
to 100 mL |
|
Stock for C buf
| 1 M HEPES-KOH (pH 7.9) |
2 mL |
20 mM |
| Glycerol |
20 mL |
20%* |
| 5 M NaCl |
8.4 mL |
420 mM |
| 1 M MgCl2 |
150 µL |
1.5 mM |
| 0.5 M EDTA |
40 µL |
0.2 mM |
| DW |
to 100 mL |
|
*25%でも良い。
Lysis buf for immuno precipitation
| 1 M Tris-HCl (pH 7.6) |
10 mL |
20 mM |
| 5 M NaCl |
15 mL |
150 mM |
| 10% NP40 |
50 mL |
1.0% |
| 0.5 M EDTA |
1 mL |
1 mM |
| DW |
to 500 mL |
|
Store at 4°C. Add protease inhibitors before use.
NP40の終濃度は0.01-1%まで実験条件に合わせて調整する。
RIPA buf
| 1 M Tris-HCl (pH 8.0) |
5 mL |
50 mM |
|
| 5 M NaCl |
3 mL |
150 mM |
| 0.5 M EDTA |
0.4 mL |
2 mM |
|
| 10% NP40 |
1 mL |
1.0% |
|
| 10% SDS |
1 mL |
1.0% |
|
| Sodium deoxycholate |
0.5 g |
0.5% |
Wako, 192-08312 |
| DW |
to 100 mL |
|
|
store at 4°C. Add protein inhibitors before use.
可溶化は強力だが、変性作用も強い。目的に応じて他のlysis bufと使い分ける。
Adding protein inhibitors
Lysis buf for whole cell lysate
| 1 M Tris-HCl (pH 7.4) |
10 mL |
100 mM |
| 5 M NaCl |
1.5 mL |
75 mM |
| Triton-X 100 |
1 mL |
(.0% |
| DW |
to 100 mL |
|
store at 4°C. Add protein inhibitors before use.
Adding 1% NP40 might improve the effeciency of nuclear protein extraction.
100×protease inhibitors
| PMSF (MW 174) |
0.87 g |
100 mM |
| Pepstatin A (MW 686) |
6.85 mg |
0.2 mM |
| Leupeptin (MW 427) |
1.42 mg |
60 µM |
| Chymostatin (MW 608) |
10 mg |
0.2 mg/mL |
| Benzamidine (MW 165) |
1.65 g |
200 mM |
| EtOH |
to 50 mL |
|
Store at -20°C.
1 M NaF
| NaF (MW 42) |
4.2 g |
Wako, 192-01972 |
| DW |
to 100 mL |
|
Store at 4°C.
50 mM sodium orthovanadate (Na3VO4)
| Sodium orthovanadate (MW 184) |
920 mg |
Wako, 198-09752 |
| DW |
to 100 mL |
|
Store at 4°C.
1 M sodium butyrate
| Sodium butyrate (MW 110) |
1.1 g |
Wako, 193-01522 |
| DW |
to 10 mL |
|
Store at 4°C. Add to cell lysate (final 10 mM). Gu, Cell (1997), 90: 595.
500 mM DTT ((±)-Dithiothreitol)
| DTT (MW 154) |
385.6 mg |
Wako, 045-08974 |
| DW |
to 5 mL |
|
Dispense and store at -20°C.
1 N glacial acetic acid for peptide extraction
| Glacial acetic acid |
31.5 mL |
| Triton-X 100 |
0.5 mL |
| DW |
to 500 mL |
10×Transfer buf [New]
| Glycine |
116.7 g |
| Tris |
24.3 g |
| DW |
to 1000 mL |
5×Transfer buf [Old]
| Glycine |
14.5 g |
| Tris |
29.0 g |
| SDS |
1.85 g |
| DW |
to 1000 mL |
10×Running buf (Tris-glycine electrophoresis buf) [Laemmli method]
| Glycine |
144 g |
192 mM@×1 |
| Tris |
30 g |
25 mM@×1 |
| SDS |
10 g |
0.1%@×1 |
| DW |
to 1000 mL |
|
5×Running buf (Tris-glycine electrophoresis buf) [Old]
| Glycine |
94 g |
| Tris |
15.1 g |
| 10% SDS |
50 mL |
| DW |
to 1000 mL |
10% SDS
5×sample buf for Western (gel-loading buf)
| 0.5 M Tris-HCl (pH 6.8) |
10 mL |
| Glycerol |
10 mL |
| SDS |
2 g |
| Bromophenol blue |
0.1 g |
| DW |
total 20 mL |
Wash buf for Western (0.5% PBS-T)
| PBS or TBS |
995 mL |
| 10% Tween20 |
5 mL |
SDS (例：0.1%) やNP40 (例：1%) を加えてwashの程度を強くしても良い。
Stripping buf
| 10% SDS |
100 mL |
| 1 M Tris-HCl (pH 6.7) |
31.25 mL |
| DW |
to 500 mL |
Add 2-ME 7 µL/stripping buf 1 mL. To strip membrane, incubate at 50-60°C for 30 min.
Stripping buf II
| Glycine |
1.5 g |
0.4 M |
| 10% SDS |
1 mL |
0.2% |
| Twee20 |
1 mL |
2% |
| 6 M HCl |
2 mL |
to pH 2.0-2.5 |
| DW |
to 50 mL |
|
Prepare before use. To strip membrane, incubate at RT for 30 min twice.
10% APS (anmonium persulfate)
Store at 4°C in the dark.
1.5 M Tris-HCl
| Tris |
54.5 g |
| HCl |
Adjust pH |
| DW |
to 300 mL |
Adjust pH at RT, store at 4°C (or RT).
0.5 M Tris-HCl
| Tris |
12.0 g |
| HCl |
Adjust pH |
| DW |
to 200 mL |
Adjust pH at RT, store at 4°C (or RT).
50 mM TBS for DAB tablet
| 1 M Tris-HCl (pH 8.0) |
50 mL |
50 mM |
| 5 M NaCl |
30 mL |
150 mM |
| DW |
to 1000 mL |
|
Geneticin disulfate (Neomycin)
100 mg/mL in PBS (10-20 mL)
Filteration by 0.22 µm filter.
Store at 4°C (in the dark?)
ACK lysis buf for hemolyzing erythrocytes or separating leukocytes
| NH4Cl |
8.29 g |
0.15 M |
| KHCO3 |
1 g |
1.0 mM |
| EDTA-2Na |
37.2 mg |
0.1 mM |
| DW |
800 mL |
|
| HCl or NaOH |
to pH 7.3 |
|
| DW |
to 1000 mL |
|
Store at 4°C.
Old-prepared ACK buf cannot hemolyze erythrocytes. Should be re-prepared.
For separating leukocytes, add 10-20 times vol of ACK buf (much is better) to blood and centrifuge immediately (with in a few minitues).
SNET buf for genomic DNA extraction from mice tails
| 1 M Tris-Cl (pH8.0) |
1.0 mL |
20 mM |
| 0.5 M EDTA |
0.5 mL |
5 mM |
| NaCl |
1.17 g |
400 mM |
| SDS |
0.5 g |
1.0% |
| DW |
to 50 mL |
|
SNET buf [Illustrated]
| 1 M Tris-Cl (pH8.0) |
0.5 mL |
10 mM |
| 0.5 M EDTA |
1.0 mL |
10 mM |
| 5 M NaCl |
1.5 mL |
150 mM |
| 10% SDS |
0.5 mL |
0.1% |
| DW |
to 50 mL |
|
5×Stock for EMSA
| 0.5 M EDTA |
500 µL |
| 1 M Tris |
1 mL |
| 3 M KCl |
3.125 mL |
| DW |
to 50 mL |
Store at 4°C.