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.
- 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
- Tris — 58g
- Glycine — 29g
- SDS — 3g
- H2O — 10 L
- 5% NFDM/TBST
- NaCl — 1314.9 g
- Tris — 545.3 g
- H2O — 22.5 L
- 10X TBS — 2.25 L
- Tween-20 — 22.5 mL
- H2O - 20.25 L
- 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
- H2O — 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.
- Load 20 μg of total protein per lane.
Run the gel for ~1.5 h at 150 V, referring to the molecular weight markers and using the pre-chilled running buffer.
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.