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Western blot coomassie blue stain

Western Blot protocol for high molecular weight proteins (150 – 300 kDa)

The following protocol is suitable for performing a Western Blot on ​​larger proteins, 150 – 300 kDa. 


For most standard sizes, see our general Western Blot protocol.


Solutions & reagents

Prepare solutions, reagents and the gels according to the recipes below.




10 x running buffer
  • Tris  — 151.425 g
  • Glycine — 720.67 g
  • SDS —​ 50 g (1 %) 
  • H2O — 5 L

Dissolve compoments in 3.5 L of water initially, then make up to 5 L

Dilute 1:10 in to 1X when ready for use

1X transfer buffer
  • Tris — 58g
  • Glycine — 29g
  • SDS — 3g
  • H2O — 10 L

Dissolve compoments in 3.5 L of water initially, then make up to 10 L

Add 10 % methanol to transfer buffer just before use

Blocking buffer
  • 5% NFDM/TBST

10X TBS
  • NaCl — 1314.9 g
  • Tris — 545.3 g
  • H2O — 22.5 L
Dissolve in deionized water, adjust pH to 7.4 using HCl, then make up to 22.5 L with deionized water. 
1X TBST
  • 10X TBS — ​2.25 L
  • Tween-20 — 22.5 mL
  • H2O - 20.025 L
Add 2.25 L 10X TBS and 22.5 L Tween-20, make up to 22.5 L with deionized water
Separation gel
  • H2O — 4.24 mL
  • 1.5 M Tris-HCl, pH 8.8 — ​2.0 mL
  • 30 % Acr/Bis — 1.6 mL
  • 10 % SDS — 80 µL
  • 10 % APS — 80 µL
  • TEMED — 5 µL

​
Stacking gel
  • H​2O — 1.32 mL
  • 1.5 M Tris-HCl, pH 6.8 — ​0.55 mL
  • 30 % Acr/Bis — 0.295 mL
  • 10 % SDS — 22 µL
  • 10 % APS — 11 µL
  • TEMED — 2.2 µL


Method


Loading and running the gel

  1. Load equal amounts of protein into the wells of the SDS-PAGE gel, along with molecular weight marker. Load 20 μg of total protein per lane.
  2. Run the gel for ~1.5 h at 150 V, referring to the molecular weight markers and using pre-chilled running buffer.

The time and voltage may require optimization. We recommend following the manufacturer’s instructions. A reducing gel should be used unless non-reducing conditions are recommended on the antibody datasheet.


Transferring the gel from the plate to the membrane

​

  1. Immerse the gel in 1× transfer buffer for 40 min.
  2. Activate the PVDF membrane with 99.5% methanol for 15 seconds.
  3. Immerse PVDF membrane, filter paper and sponge in 1× transfer buffer for 30 min before transfer.
  4. Complete a wet transfer at 500 mA, for 1h, at 4°C using pre-chilled transfer buffer.
  5. Once complete, wash twice for 10 minutes in deionized water, before drying and storing at 4°C or continuing with antibody staining.


Antibody staining​

  1. Block the membrane for 1 h at room temperature or overnight at 4°C using 5 % blocking buffer.
  2. Wash the membrane with 1x TBST for 10 minutes.
  3. Incubate the membrane with appropriate dilutions of primary antibody in blocking buffer for 1 hour at room temperature on a shaker with a low setting.
  4. Wash the membrane in three washes of TBST, 10 min each.
  5. Incubate the membrane with the recommended dilution of conjugated secondary antibody in blocking buffer at room temperature for 1 h on a shaker with a low setting.
  6. Wash the membrane in three washes of TBST, 10 min each.
  7. For signal development, follow the kit manufacturer’s recommendations. Remove excess reagent and cover the membrane in transparent plastic wrap.
  8. Acquire image using darkroom development techniques for chemiluminescence, or normal image scanning methods for colorimetric detection.