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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

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

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

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

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

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

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

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

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Stage 3 - Antibody staining

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

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Wash the membrane with 1x TBST for 10 minutes.

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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.

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

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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.

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

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

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

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