Western Blot Buffers & Recipes

Western Blot Buffers and Recipes

Find below recipes for all essential western blotting buffers including: blocking buffer, lammeli buffer, loading buffer, running buffer, transfer buffer and stripping buffer. For a complete guide to western blotting click here

Blocking Buffer

  • 5% Marvel in TBST

Lameli Buffer Recipe

  • 4% SDS
  • 10% 2-mercaptoehtanol
  • 20% glycerol,
  • 0.004% bromophenol blue,
  • 0.125 M Tris HCl.
  • Check the pH and bring it to pH 6.8.

Loading Buffer Recipe

  • 2X Loading buffer:
  • 100 mM Tris-HCL (pH6.8)
  • 200 mM DTT
  • 4% SDS
  • 0.2% Bromophenol blue
  • 20% glycerol
  • (DTT should be added just before the buffer is used, from 1M stock)

NP-40 Buffer Recipe

  • 150 mM sodium chloride,
  • 1.0% NP-40 (Triton X-100 can be substituted for NP-40),
  • 50 mM Tris pH 8.0

Resolving Gel Recipe

  • H20 5.9 ml
  • 30% Acrylamide-Bis 5 ml
  • 1.5M Tris pH 8.8 3.8 ml
  • 10% SDS 150 µl
  • 10% APS 150 µl
  • Temed 6 µl

RIPA Buffer Recipe

  • 50mM Tris HCL pH 7.4,
  • 50 mM NaCl,
  • 2mM EDTA,
  • 1% SDS.
  • Plus freshly added proteinase Inhibitors (Apoprotein, Leupeptin, DTT and PMSF)

Running Buffer Recipe

  • 15.1 g TRIZMA,
  • 94 g glycine
  • 50 ml of 10% w/v SDS,
  • dH20 to 1 litre

Tris-HCL Buffer

  • 20 mM Tris-HCl, pH 7.

Tris-Triton Buffer

  • 10 mM Tris, pH 7.4
  • 100 mM NaCl
  • 1 mM EDTA
  • 1 mM EGTA
  • 1% Triton X-100
  • 10% glycerol
  • 1% SDS
  • 5% deoxycholate

Stacking Gel Recipe

  • H20 2.7 ml
  • Acrylamide-Bis 670 µl
  • Tris pH 6.8 500 µl
  • 10% SDS 40 µl
  • 10% APS 40 µl
  • Temed 3 µl

Stripping Buffer

  • 62.5 mM Tris-HCL,pH 7.8,
  • 100 mM -mercaptoethanol,
  • 2% (w/v) SDS

TBS-T Recipe

  • 20 mM Tris, pH 7.5
  • 150 mM NaCl
  • 01% Tween 20

Transfer Buffer Recipe

  • 8g Trizma base
  • 9g glycine
  • 37g SDS powder
  • 200ml methanol
  • dH20 to 1L