Chapter 5: Western Blot Buffers

The following buffers and methods provide a general starting point for use with the majority of Bio-Rad reagents in Western blotting . When specific recommendations are provided on product datasheets, those instructions should always be used instead of the general guidance offered here . With specialized laboratory equipment, such as for gel electrophoresis and electroblotting, it is advisable to follow the manufacturer’s instructions .

If difficulties arise with any of our antibodies or related products, please feel free to contact antibody_tech_us@bio-rad.com as we may have additional information on file and can assist with troubleshooting.

Buffers

N .B . Azide should not be added to any buffers that will be used with HrP-labeled antibodies because it will inactivate the HrP enzyme.

PBS

  • 8 .0 g NaCl
  • 0 .2 g KCl
  • 1 .15 g Na2HPO4
  • 0 .2 g KH2PO4
  • Dissolve in 800 ml distilled water, adjust pH to 7 .4, and then add more dH20 to a final volume of 1 liter . Sterilize by autoclaving and store at room temperature (RT).

TBS

  • 8 .0 g NaCl
  • 0 .2 g KCl
  • 3 .0 g Tris base
  • Dissolve in 800 ml distilled water, adjust pH to 8 .0 with 1 M HCl, and then add more dH20 to a final volume of 1 liter. Sterilize by autoclaving and store at RT.

10x TBS Stock:

  • 500 mM Tris-HCl, pH 7 .4
  • 1 .5 M NaCl

 

Cell Lysis Buffers

NP-40 Lysis Buffer

  • 50 mM Tris, pH 8 .0
  • 150 mM NaCl
  • 1% NP-40 (or Triton® X-100)
  • + fresh protease inhibitors, see below

RIPA (Radio Immuno Precipitation Assay) Buffer

  • 50 mM Tris, pH 8 .0
  • 150 mM NaCl
  • 1% NP-40 (or Triton® X-100)
  • 0 .5% Sodium deoxycholate
  • 0 .1% SDS
  • + fresh protease inhibitors, see below

 

Proteases Inhibitors

Various proteases inhibitors are often added to lysis buffer to maintain the integrity of the target protein when cells are disrupted. The following table provides some recommended working concentrations.

Inhibitor Working Concentration Protease Inhibited
Aprotinin 1-2 µg/ml
Trypsin, Chymotrypsin, 
Plasmin, Kallikrein
EDTA 1-2 mM
Metalloproteases (Mg2
and Mn2+)
EGTA 1 mM Metalloproteases (Ca2+)
Leupeptin 0 .5-2 µg/m
Plasmin, Trypsin, Papain, 
Cathepsin B
Pepstatin A 1 µg/ml Pepsin, Cathepsin D
PMSF (highly toxic) 50-100 µg/ml
Serine and Cysteine 
proteases

 

Electrophoresis and Transfer Buffers

Laemmli 2x Sample Buffer

  • 4% SDS
  • 20% Glycerol 125 mM Tris, pH 6 .8
  • 0 .02% Bromophenol blue 200 mM DTT or 10% ßME
  • For best results DTT or ßME is added fresh, just before use.

Gel Electrophoresis Running Buffer

  • 25 mM Tris base
  • 190 mM Glycine
  • 0 .1% SDS

Transfer Buffer

  • 50 mM Tris base
  • 380 mM Glycine
  • 0 .1% SDS
  • 20% Methanol

Ponceau S Stock Solution

  • 2% Ponceau S 
  • 30% Trichloroacetic acid
  • 30% Sulfosalicylic acid
  • Dilute 10-fold in distilled water prior to use

 

Washing Buffers

PBST 

  • PBS with 0 .1% Tween® 20

TBST

  • TBS with 0 .1% Tween® 20

Block Ace Wash Buffer (BUF029) 

Reconstitute each 4 g vial in 100 ml distilled water . Dilute the reconstituted solution 10-fold, and add Tween® 20 to a final concentration of 0 .05-0 .2% v/v.


 

Blocking Buffers

*Use BSA or Block Ace (BUF029) to block when probing with anti-phosphoprotein antibodies, or for biotinylated primary antibodies detected with an anti-biotin secondary.

5% Nonfat Dried Milk in PBST or TBST (Blotto/BLOTTO)

  • Add 5 g nonfat dried milk powder to 100 ml PBST or TBST. 
  • Dissolve with gentle stirring . Store at 4°C. 

3% BSA in PBST or TBST 

  • Dissolve 3 g of BSA Fraction V in 100 ml PBST or TBST with gentle stirring. 
  • Store at 4°C.

Block Ace (BUF029) 

  • Reconstitute each 4 g vial in 100 ml distilled water, and use undiluted.

 

 


Chapter 4: Alternate Forms of a Protein - Prion Disease Western Blot Protocol: Cell Lysis, Mammalian Cells