Buffers and stock solutions for western blot

Lysis buffers differ in their ability to solubilize proteins. The buffers containing sodium dodecyl sulfate (SDS) and other ionic detergents are the most efficient in extracting membrane proteins from lipid bilayers at a high yield.

When choosing a lysis buffer, the main consideration is whether the selected antibody will recognize denatured samples. When this is not the case, it will be noted on the antibody datasheet, and buffers without detergent or with relatively mild non-ionic detergents (NP-40, Triton X-100) should be used. 

Protein location and lysis buffer choice

*Proteins found exclusively or predominantly in a subcellular location will be more enriched in a subcellular fraction lysate than whole cell or tissue lysates. This can be useful when obtaining a signal for a weakly-expressed protein. Please consult our separate protocols for subcellular fractionation.

All four of these buffers below can be kept at 4°C for several weeks or for up to a year if divided into aliquots and stored at -20°C.

NP-40 is a popular buffer for studying cytoplasmic and membrane-bound proteins and for whole-cell extracts. Suppose you are concerned that the protein of interest is not being completely extracted from insoluble material or aggregates. In that case, RIPA buffer may be more suitable as it contains ionic detergents that will solubilize proteins more efficiently.

• 150 mM NaCl
• 1.0% NP-40 (possible to substitute with 0.1% Triton X-100)
• 50 mM Tris-HCl, pH 8.0
• Protease inhibitors

RIPA buffer contains the ionic detergent sodium deoxycholate as an active constituent and is useful for lysis of whole-cell extracts and membrane-bound proteins. Also, RIPA buffer may be preferable to NP-40 or Triton X-100-only buffers for extracting nuclear proteins.

A RIPA buffer will disrupt protein-protein interactions and may, therefore, be problematic for immunoprecipitation (IP) and pull-down assays prior to western blot. When it’s crucial to preserve protein-protein interactions or to minimize denaturation, you should use a buffer without ionic detergents (eg SDS) and ideally without non-ionic detergents (eg Triton X-100).

Cell lysis with detergent-free buffer is achieved by mechanical shearing, often with a Dounce homogenizer or by passing cells through a syringe needle. In these cases, a simple Tris buffer will suffice, but as noted above, buffers with detergents are required to release membrane- or cytoskeleton-bound proteins.

• 50 mM Tris-HCl, pH 8.0
• 150 mM NaCl
• 1% NP-40
• 0.5% sodium deoxycholate - can be prepared as a 10% sodium deoxycholate stock solution (5 g into 50 mL), which must be protected from light.
• 0.1% SDS (sodium dodecyl sulfate)
• Protease inhibitors
• Phosphatase inhibitors – if the samples will be used for IP before proceeding with western blot. Note that using phosphatase inhibitors will interfere with phosphatase treatment if this is required before WB.
• 1mM EDTA – optional*

*Some RIPA buffer recipes include 1mM EDTA – a chelator of divalent cations (an important cofactor of many enzymes, such as DNases and proteases) – to inhibit proteases, help dissociate ribosomal subunits and protein-RNA complexes, etc.

• 20 mM Tris-HCl, pH 7.5
• Protease inhibitors

• 10 mM Tris, pH 7.4
• 100 mM NaCl
• 1 mM EDTA
• 1 mM EGTA
• 1 mM NaF
• ​20 mM Na₄P₂O₇
• 2 mM Na₃VO₄
• 1% Triton X-100
• 10% glycerol
• 0.1% SDS
• 0.5% deoxycholate

As soon as lysis occurs, proteolysis, dephosphorylation, and denaturation begin. These events can be slowed down significantly if samples are kept on ice or at 4°C at all times, and appropriate inhibitors are added fresh to the lysis buffer.

Ready-to-use cocktails of inhibitors are available from various suppliers, but you can prepare your own inhibitor cocktail.

Perform all the steps under the fume hood.

1. Prepare a 100 mM sodium orthovanadate solution with double distilled water.
2. Set pH to 9.0 with HCl.
3. Boil until colorless*.
4. Cool to room temperature. Minimize volume change due to evaporation by covering loosely.
5. Set pH to 9.0 again.
6. Boil again until colorless*.
7. Repeat this cycle until the solution remains at pH 9.0 after boiling and cooling*.
8. Bring up to the initial volume with water.
9. Store in aliquots at -20°C.
10. Discard if the samples turn yellow.

*Avoid large changes in volume during boiling; put a loose lid on the container to protect it from evaporation.

• 20 mM Tris-HCI, pH 7.5
• 1 mM EGTA (Ca²⁺ chelator)

Loading buffer/Laemmli 2X buffer

• 4% SDS
• 10% 2-mercaptoethanol
• 20% glycerol
• 0.004% bromophenol blue
• 0.125 M Tris-HCl
• Check the pH and adjust it to 6.8

Running buffer (Tris-Glycine/SDS)

• 25 mM Tris base
• 190 mM glycine
•  0.1% SDS
• Check the pH and adjust to 8.3

Transfer buffer (wet)

• 25 mM Tris base
• 190 mM glycine
•  20% methanol
• Check the pH and adjust to 8.3
• For proteins >80 kDa, we recommend including SDS at a final concentration of 0.1%.

Transfer buffer (semi-dry)

• 48 mM Tris
• 39 mM glycine
• 20% methanol
• 0.04% SDS

Blocking buffer

• 3–5% milk or BSA (bovine serum albumin)
• Add BSA to Tris-buffered saline (TBS) or TBST (TBS containing  0.1% Tween 20). 
• Mix well and filter. Failure to filter can lead to spotting, with tiny dark dots contaminating the blot during signal development and interfering with protein band visualization. 

This 10X TBS stock solution contains 200 mM Tris and 1500 mM NaCl.

For 1 L:

• 24 g Tris base (formula weight: 121.1 g)
• 88 g NaCl (formula weight: 58.4 g)
• Dissolve in 900 mL of distilled water
• pH to 7.6 with 12 N HCl
• Add distilled water to a final volume of 1 L

For a 1x solution, mix 1 part of the 10x solution with 9 parts distilled water and adjust pH to 7.6 again. The final molar concentrations of the 1x solution are 20 mM Tris and 150 mM NaCl. 

An alternative recipe for Tris buffer combines Tris base and Tris-HCl. This avoids the large volume of potentially hazardous hydrochloric acid needed to neutralize a Tris base solution alone.

For 1 L:

• 24 g Tris-HCl (formula weight: 157.6 g)
• 5.6 g Tris base (formula weight: 121.1 g)
• 88 g NaCl (formula weight: 58.4 g)
• Dissolve in 900 mL of distilled water

1. The pH of the solution should be about 7.6 at room temperature. If too basic, adjust to pH 7.6 with concentrated HCl; if too acidic, adjust with concentrated NaOH.
2. Add distilled water to a final volume of 1 L.
3. For a 1x solution, mix 1 part 10x with 9 parts distilled water and pH to 7.6 again.

The final molar concentrations of the 1x solution are 20 mM Tris and 150 mM NaCl.

For 1 L:

• 100 mL of TBS 10x
• 900 mL distilled water
• 1 mL Tween 20*

*Tween 20 is very viscous and will stick to the tip of your measuring pipettes. Be sure you add the right amount of the detergent to the Tris buffer. A 10% solution is easier to dispense than undiluted Tween 20. 

• 15 g glycine
• 1 g SDS
• 10 mL Tween 20

1. Adjust the volume to 800 mL with distilled water.
2. Adjust pH to 2.2.
3. Bring volume up to 1 L with distilled water.

Prepare buffer and strip membranes under a fume hood.

For 100 mL:

• 20 mL SDS 10%
• 12.5 mL Tris HCl, pH 6.8, 0.5 M
• 67.5 mL distilled water
• ​Add 0.8 mL β-mercaptoethanol under the fume hood

The following nuclear fractionation buffers are used for extracting and fractionating the nuclear fraction of cells. The main difference between the buffer recipes below is that buffer A contains a detergent, NP-40.

• 10 mM HEPES
• 1.5 mM MgCl₂
• 10 mM KCl
• 0.5 DTT
• ​0.05% NP-40 (or 0.05% Igepal or Tergitol) pH 7.9

To prepare 250 mL stock of buffer A:

• HEPES: 1 M = 238.3 g/L, therefore 10 mM = 0.59 g/250 mL
• MgCl₂: 1 M = 203.3 g/L, therefore 1.5 mM = 0.076 g/250 mL
• KCl: 1 M = 74.5 g/L, therefore 10 mM = 0.187 g/250 mL
• DTT: 1 M = 154.2 g/L, therefore 0.5 mM = 0.019 g/250 mL
• NP-40: 0.05%

• 5 mM HEPES
• 1.5 mM MgCl₂
• ​0.2 mM EDTA
• 0.5 mM DTT
• 26% glycerol (v/v) pH 7.9

To prepare 250 mL stock of buffer B:

• HEPES: 1 M = 238.3 g/L, therefore 5 mM = 0.295 g/250 mL
• MgCl₂: 1 M = 203.3 g/L, therefore 1.5 mM = 0.076 g/250 mL
• EDTA: 1 M = 372.2 g/L, therefore 0.2 mM = 0.0186 g/250 mL
• DTT: 1 M = 154.2 g/L, therefore 0.5 mM = 0.019 g/250 mL
• ​26% glycerol (v/v) = 65 mL

Example of 10 mL primary antibody solution containing any primary antibody at 1:1000 dilution:

• 10 µL primary antibody
• 0.1 g BSA
• 9.90 mL 1X TBS, pH 7.6–7.8

An example of ABC solution, with each part used at a dilution of 1:100.

For 1 mL:

• 10 µL Streptavidin
• ​10 µL HRP (or AP)-biotin
• 980 µL 1X TBS pH 7.6–7.8

• 3.03 g Na₂CO₃
• 6.0 g NaHCO_​3 (1 L distilled water) pH 9.6