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Western blot for high molecular weights

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.

Last edited Tue 02 May 2023

Solutions & reagents

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

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

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

Dilute 1:10 into 1X when ready for use

1X transfer buffer
  • Tris — 58g
  • Glycine — 29g
  • SDS — 3g
  • H2O — 10 L
Dissolve in deionized water, adjust pH to 7.4 using HCl, then make it up to 22.5 L with deionized water.
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 it up to 22.5 L with deionized water.
1X TBST
  • 10X TBS — ​2.25 L
  • Tween-20 — 22.5 mL
  • H2O - 20.25 L
Add 2.25 L 10X TBS and 22.5 mL 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

Stage 1 - Loading and running the gel

Steps

Load equal amounts of protein into the wells of the SDS-PAGE gel, along with the molecular weight marker.

Run the gel for ~1.5 h at 150 V, referring to the molecular weight markers and using the 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.

Stage 2 - Transferring the gel from the plate to the membrane

Steps

Immerse the gel in 1X transfer buffer for 40 min.

Activate the PVDF membrane with 99.5% methanol for 15 sec.

Immerse PVDF membrane, filter paper, and sponge in 1X transfer buffer for 30 min before transfer.

Complete a wet transfer at 500 mA, for 1 h, at 4°C using the pre-chilled transfer buffer.

Once complete, wash twice for 10 minutes in deionized water before drying and storing at 4°C or continuing with antibody staining.

Stage 3 - Antibody staining

Steps

Block the membrane for 1 h at room temperature or overnight at 4°C using 5% blocking buffer.

Wash the membrane with 1x TBST for 10 minutes.

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.

Wash the membrane in three washes of TBST, 10 min each.

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.

Wash the membrane in three washes of TBST, 10 min each.

For signal development, follow the kit manufacturer’s recommendations. Remove excess reagent and cover the membrane in transparent plastic wrap.

Acquire images using darkroom development techniques for chemiluminescence, or normal image scanning methods for colorimetric detection.