Western Blot Buffer Recipes
Stock solutions
- 1 M Tris-HCl, pH 7.6 (100 mL)
- 0.5 M Tris-HCl, pH 6.8 (100 mL)
- 10% SDS (10 mL)
- 1.0% Bromophenol Blue (10 mL)
- 10X Tris Buffered Saline (TBS)
- 10X Phosphate Buffered Saline (PBS)
Sample preparation buffers
- RIPA buffer
- 2X Tris-Glycine SDS Sample buffer (Laemmli buffer)
- 4X LDS Sample Buffer
Electrophoresis running buffers
- 10X Tris-Glycine SDS Running Buffer
- 10X Tris-Glycine Native Running Buffer
- 20X MOPS SDS Running Buffer
- 20X MES SDS Running Buffer
- 10X Tricine SDS Running Buffer
Transfer buffer
- 25X Tris-Glycine Transfer Buffer
- 20X Bis-Tris Transfer Buffer
Wash buffers
- Tris-buffered saline with Tween 20 (TBST)
- Phosphate buffered saline with Tween 20 (PBST)
Blocking and stripping buffers recipes
- 5% Nonfat Milk
- 3% BSA
- Stripping Buffer
Gel casting recipes
- SureCast Reagents
- Standalone Reagents
Stock solutions
1 M Tris-HCl, pH 7.6 (100 mL)
| Tris Base | 12.11 g |
| Deionized water | 80 mL |
| Adjust pH to 7.6 with HCl | |
| Deionized water | to 100 mL |
0.5 M Tris-HCl, pH 6.8 (100 mL)
| Tris Base | 6.06 g |
| Deionized water | 60 mL |
| Adjust pH to 6.8 with HCl | |
| Deionized water | to 100 mL |
10% SDS (10 mL)
| SDS | 1.00 g |
| Deionized water | to 10 mL |
1.0% Bromophenol Blue (10 mL)
| Bromophenol blue | 100 mg |
| Deionized water | to 10 mL |
10X Tris Buffered Saline (TBS)
| Tris Base | 24 g |
| NaCl | 88 g |
| Deionized water | 900 mL |
| pH to 7.6 with HCl | |
| Deionized water | to 1000 mL |
Ready-to-use alternative:
Pierce 20X TBS Buffer, 500 mL (Cat. No. 28358)
10X Phosphate Buffered Saline (PBS)
| NaCl | 80 g |
| KCl | 2 g |
| Na2HPO4 | 14.4 g |
| NaH2PO4 | 2.4 g |
| Deionized water | 900 mL |
| pH to 7.0 with NaOH | |
| Deionized water | to 1000 mL |
Ready-to-use alternative:
Pierce 20X PBS Buffer, 500 mL (Cat. No. 28348)
Sample preparation buffers
RIPA buffer: 25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS (100 mL)
| NaCl | 0.88 g |
| NP-40 | 1 g |
| Sodium deoxycholate | 1 g |
| 10% SDS | 1 mL |
| 1 M Tris-HCl, pH 7.6 | 2.5 mL |
| Deionized water | to 100 mL |
| Protease Inhibitor Tablet (Cat. No. A32965) | 2 tablets |
Ready-to-use alternative:
Thermo Scientific RIPA Lysis and Extraction Buffer, 100 mL (Cat. No. 89900)
SDS Sample buffer (Laemmli buffer): 63 mM Tris HCl, 10% Glycerol, 2% SDS, 0.0025% Bromophenol Blue, pH 6.8 (10 mL)
Recipe for 2X buffer stock:
| 0.5 M Tris-HCl pH 6.8 | 2.5 mL |
| Glycerol | 2 mL |
| 10% (w/v) SDS | 4 mL |
| 0.1% (w/v) Bromophenol Blue | 0.5 mL |
| Deionized water | to 10 mL |
The buffer is stable for 6 months when stored at 4°C.
Ready-to-use alternative:
Invitrogen Novex Tris-Glycine SDS Sample Buffer (2X) (Cat. No. LC2676)
LDS Sample Buffer: 106 mM Tris HCl, 141 mM Tris Base, 2% LDS, 10% Glycerol, 0.51 mM EDTA, 0.22 mM SERVA Blue G250, 0.175 mM Phenol Red, pH 8.5.
Recipe for 4X buffer stock:
| Tris HCl | 0.666 g |
| Tris Base | 0.682 g |
| LDS | 0.800 g |
| EDTA | 0.006 g |
| Glycerol | 4 g |
| SERVA Blue G250 (1% solution) | 0.75 mL |
| Phenol Red (1% solution) | 0.25 mL |
| Deionized water | to 10 mL |
The buffer is stable for 6 months when stored at 4°C. Do not use acid or base to adjust pH.
Ready-to-use alternative:
Invitrogen NuPAGE LDS Sample Buffer (4X) (Cat. No. NP0007)
Electrophoresis running buffers
Tris-Glycine SDS Running Buffer: 25 mM Tris Base, 192 mM Glycine, 0.1% SDS, pH 8.3.
Recipe for 10X buffer stock:
| Tris Base | 29 g |
| Glycine | 144 g |
| SDS | 10 g |
| Deionized water | to 1000 mL |
Ready-to-use alternative:
Novex Tris-Glycine SDS Running Buffer (10X), 500 mL (Cat. No. LC2675)
Tris-Glycine Native Running Buffer: 25 mM Tris Base, 192 mM Glycine, pH 8.3.
Recipe for 10X buffer stock:
| Tris Base | 29 g |
| Glycine | 144 g |
| Deionized water | to 1000 mL |
Ready-to-use alternative:
Novex Tris-Glycine Native Running Buffer (10X), 500 mL, 500 mL (Cat. No. LC2672)
MOPS SDS Running Buffer: 50 mM MOPS, 50 mM Tris Base, 0.1% SDS, 1 mM EDTA, pH 7.7.
Recipe for 20X buffer stock:
| MOPS | 104.6 g |
| Tris Base | 60.6 g |
| SDS | 10 g |
| EDTA | 3.0 g |
| Deionized water | to 500 mL |
Do not use acid or base to adjust pH.
Ready-to-use alternative:
NuPAGE MOPS SDS Running Buffer (20X), 500 mL (Cat. No. NP0001)
MES SDS Running Buffer: 50 mM MES, 50 mM Tris Base, 0.1% SDS, 1 mM EDTA, pH 7.3.
Recipe for 20X buffer stock:
| MES | 97.6 g |
| Tris Base | 60.6 g |
| SDS | 10 g |
| EDTA | 3.0 g |
| Deionized water | to 500 mL |
Do not use acid or base to adjust pH.
Ready-to-use alternative:
NuPAGE MES SDS Running Buffer (20X), 500 mL (Cat. No. NP0002)
Tricine SDS Running Buffer: 100 mM Tris Base, 100 mM Tricine, 0.1% SDS, pH 8.3.
Recipe for 10X buffer stock:
| Tris Base | 121 g |
| Tricine | 179 g |
| SDS | 10 g |
| Deionized water | to 1000 mL |
The buffer is stable for 6 months when stored at room temperature.
Do not use acid or base to adjust pH.
Ready-to-use alternative:
Novex Tricine SDS Running Buffer (10X), 500 mL (Cat. No. LC1675)
Transfer buffer recipes
Tris-Glycine Transfer Buffer: 12 mM Tris Base, 96 mM Glycine, pH 8.3.
Recipe for 25X buffer stock:
| Tris Base | 18.2 g |
| Glycine | 90 g |
| Deionized water | to 500 mL |
Ready-to-use alternative:
Novex Tris-Glycine Transfer Buffer (25X) 500 mL (Cat. No. LC3675)
Bis-Tris Transfer Buffer: 25 mM Bicine, 25 mM Bis-Tris (free base), 1 mM EDTA, pH 7.2.
Recipe for 20X buffer stock:
| Bicine | 10.2 g |
| Bis-Tris (free base) | 13.1 g |
| EDTA | 0.75 g |
| Deionized water | 125 mL |
The buffer is stable for 6 months when stored at 4°C.
Do not use acid or base to adjust pH.
Ready-to-use alternative:
NuPAGE Transfer Buffer (20X), 125 mL (Cat. No. NP0006)
Wash buffers recipes
Tris-buffered saline with Tween 20 (TBST)
| 10X TBS | 100 mL |
| Tween 20 | 1 mL |
| Deionized water | to 1000 mL |
Ready-to-use alternative:
Pierce 20X TBS Tween 20 Buffer, 500 mL (Cat. No. 28360)
Phosphate buffered saline with Tween 20 (PBST)
| 10X TBS | 100 mL |
| Tween 20 | 1 mL |
| Deionized water | to 1000 mL |
Ready-to-use alternative:
Pierce 20X PBS Tween 20 Buffer, 500 mL (Cat. No. 28352)
Blocking and stripping buffers recipes
5% nonfat milk
| Nonfat dry milk | 2.5 g |
| TBST or PBST | Up to 50 mL |
| Filter to remove particulates |
Ready-to-use alternative:
Pierce Clear Milk Blocking Buffer 10X, 100 mL (Cat. No. 37587)
3% BSA
| BSA | 1.5 g |
| TBST or PBST | Up to 50 mL |
| Filter to remove particulates |
Ready-to-use alternatives:
Pierce Blocker BSA (10X) in TBS, 125 mL (Cat. No. 37520)
Pierce Blocker BSA (10X) in PBS (Cat. No. 37525)
Stripping buffer
| 0.5 M Tris HCl, pH 6.8 | 12.5 mL |
| 10% SDS | 20 mL |
| 2-mercaptoethanol | 0.8 mL |
| Deionized water | 67.5 mL |
Ready-to-use alternatives:
Restore Western Blot Stripping Buffer, 500 mL (Cat. No. 21095)
Restore Fluorescent Western Blot Stripping Buffer, 100 mL (Cat. No. 62300)
Gel casting recipes
Recipes with SureCast reagents
The volumes provided in the table are for a single gel. Scale volumes proportionally based on the number of gels to be cast.
| Polyacrylamide % | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Solution | 4% | 6% | 8% | 10% | 12% | 14% | 16% | 18% | 20% |
| SureCast Acrylamide (40%) | 0.8 mL | 1.2 mL | 1.6 mL | 2.0 mL | 2.4 mL | 2.8 mL | 3.3 mL | 3.6 mL | 4.0 mL |
| SureCast Resolving Buffer | 2.0 mL | 2.0 mL | 2.0 mL | 2.0 mL | 2.0 mL | 2.0 mL | 2.0 mL | 2.0 mL | 2.0 mL |
| Distilled water | 5.1 mL | 4.7 mL | 4.3 mL | 3.9 mL | 3.5 mL | 3.1 mL | 2.7 mL | 2.3 mL | 1.9 mL |
| 10% SureCast APS | 80 µL | 80 µL | 80 µL | 80 µL | 80 µL | 80 µL | 80 µL | 80 µL | 80 µL |
| SureCast TEMED* | 8 µL | 8 µL | 8 µL | 8 µL | 8 µL | 8 µL | 8 µL | 8 µL | 8 µL |
*Add this last and mix well just before the gel is to be poured
Prepare stacking gel solution according to the following table. The volumes provided in the table are for a single gel. Scale volumes proportionally based on the number of gels to be cast. Note: Solutions do not require degassing.
| Solution | 4% |
|---|---|
| SureCast Acrylamide (40%) | 0.30 mL |
| SureCast Stacking Buffer | 0.75 mL |
| Distilled water | 1.92 mL |
| 10% SureCast APS | 30 µL |
| SureCast TEMED* | 3 µL |
*Add this last and mix well just before the gel is to be poured
Recipes with standalone reagents
Stock solutions
Prepare the following stock solutions: all solutions can be stored at room temperature.
50% Acrylamide/BIS (29:1)
Store up to two months in a dark glass bottle. | Separating Gel Buffer (1 M Tris-HCl, pH 8.8)
| Stacking Gel Buffer (0.375M Tris HCl, pH 6.8)
| Catalyst-Ammonium Persulfate (Make fresh the day of use)
|
10% SDS
| 50% Sucrose
|
Separating gel
The following recipes are for approximately 25 mL of separating gel, enough for four 1.0-mm thick mini gels. Scale volumes proportionally based on the number of gels to be cast.
| Solution | 6% Gel | 8% Gel | 10% Gel | 12% Gel | 14% Gel | 16% Gel | 18% Gel | 20% Gel |
|---|---|---|---|---|---|---|---|---|
| 50% Acrylamide/BIS | 3.0 mL | 4.0 mL | 5.0 mL | 6.0 mL | 7.0 mL | 8.0 mL | 9.0 mL | 10.0 mL |
| Separating Gel Buffer | 9.4 mL | 9.4 mL | 9.4 mL | 9.4 mL | 9.4 mL | 9.4 mL | 9.4 mL | 9.4 mL |
| 10% SDS | 250 µL | 250 µL | 250 µL | 250 µL | 250 µL | 250 µL | 250 µL | 250 µL |
| 50% Sucrose* | 4.0 mL | 4.0 mL | 4.0 mL | 4.0 mL | 4.0 mL | 4.0 mL | 4.0 mL | 4.0 mL |
| Water | 7.8 mL | 6.8 mL | 5.8 mL | 4.8 mL | 3.7 mL | 2.7 mL | 1.7 mL | 750 µL |
| TEMED** | 6.25 µL | 6.25 µL | 6.25 µL | 6.25 µL | 6.25 µL | 6.25 µL | 6.25 µL | 6.25 µL |
| Catalyst** | 625 µL | 625 µL | 625 µL | 625 µL | 625 µL | 625 µL | 625 µL | 625 µL |
*Optional but recommended because it makes it easy to form a good interface between the separating gel and the overlay. If omitted, increase the amount of water added to make up for the volume of the sucrose solution (increase the water by 4.0 mL for the above tables).
**Add these last and mix well just before the gel is to be poured.
Stacking gel
Following recipe is for 4% Stacking Gel (12.5 mL)
| Solution | 4% |
|---|---|
| 50% Acrylamide/BIS | 1.0 mL |
| Stacking Gel Buffer | 4.2 mL |
| 10% SDS | 125 µL |
| Water | 6.3 mL |
| TEMED* | 5.0 µL |
| Catalyst* | 1.0 mL |
*Add these last and mix well just before the gel is to be poured.
