Buffer recipes
Western blot buffer recipes and compatibility notes
The recipes below are common starting formulations. Gel chemistry, transfer devices and antibody protocols may require different compositions, so check them against the product instructions and laboratory SOP in use.
Safety and compatibility
Handle methanol, glacial acetic acid, SDS and other reagents under appropriate institutional controls. This page does not replace safety data sheets, training, product instructions or laboratory SOPs. Proprietary gels and rapid-transfer systems may require entirely different buffers.
1× TBST, 1 L (0.1% Tween-20)
Composition
- • 100 mL 10× TBS
- • 1.0 mL Tween-20
- • Purified water to a final volume of 1 L
Preparation
Mix TBS and most of the water first. Add Tween-20 carefully, mix until uniform, then bring to final volume.
Note: Some antibody protocols specify a different detergent concentration or PBS-based wash buffer. Follow the antibody-specific protocol when stated.
5% nonfat dry milk in TBST, 100 mL
Composition
- • 5.0 g nonfat dry milk
- • 1× TBST to a final volume of 100 mL
Preparation
Add milk gradually while stirring. Prepare it fresh enough to avoid microbial contamination and discard it if clumped, odorous or visibly contaminated.
Note: For phospho-antibodies, use the blocker or diluent validated for the specific primary antibody rather than assuming milk is always suitable or unsuitable.
5% BSA in TBST, 100 mL
Composition
- • 5.0 g BSA
- • 1× TBST to a final volume of 100 mL
Preparation
Dissolve gently to avoid foaming. Confirm that the BSA grade and buffer are compatible with the detection system.
Note: BSA improves some antibody assays and raises background in others. Use the antibody-specific recommendation when available.
1× Tris–glycine transfer buffer, 1 L with 20% methanol
Composition
- • 100 mL 10× Tris–glycine transfer buffer
- • 200 mL methanol
- • Purified water to a final volume of 1 L
Preparation
Prepare in a suitable container and cool as required by the transfer system. Handle methanol according to institutional chemical-safety rules.
Note: Large proteins may benefit from lower methanol and longer or cooler transfer, while small proteins may require a shorter transfer. Follow the device and membrane instructions.
1× Tris–glycine–SDS running buffer
Composition
- • 25 mM Tris base
- • 192 mM glycine
- • 0.1% (w/v) SDS
Preparation
Dilution from a validated concentrated stock is a reproducible way to prepare this buffer. Do not adjust pH unless the gel-system protocol explicitly requires it.
Note: This formulation is for conventional Tris–glycine SDS-PAGE and is not interchangeable with every Bis-Tris or proprietary gel system.
RIPA lysis buffer: common composition
Composition
- • 25 mM Tris-HCl, pH 7.6
- • 150 mM NaCl
- • 1% NP-40 or equivalent nonionic detergent
- • 1% sodium deoxycholate
- • 0.1% SDS
Preparation
Use a validated commercial or institutional formulation when possible. Add protease and, when needed, phosphatase inhibitors immediately before use.
Note: RIPA is relatively stringent but does not fully solubilize every membrane, cytoskeletal or aggregated target. Check recovery for the protein being measured.
0.1% Ponceau S in 5% acetic acid, 100 mL
Composition
- • 0.10 g Ponceau S
- • 5 mL glacial acetic acid
- • Purified water to a final volume of 100 mL
Preparation
Dissolve completely and filter if particulate material is present. Stain briefly, document the whole membrane, then destain with water or the method specified by your SOP.
Note: Ponceau S is useful for a quick transfer check but has limited sensitivity. If it is used for quantification, confirm that the method is suitable for that purpose.
Label every buffer with
Name, concentration, pH when applicable, preparation date, preparer, storage conditions, inhibitor-addition time and any change from the standard formulation. Old or contaminated buffers can cause speckles and high background.