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 |
| 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 |
| 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 |
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| 10X TBS |
| Dissolve in deionized water, adjust pH to 7.4 using HCl, then make up to 22.5 L with deionized water. |
| 1X TBST |
| Add 2.25 L 10X TBS and 22.5 L Tween-20, make up to 22.5 L with deionized water |
| Separation gel |
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| Stacking gel |
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Method
Loading and running the gel
- 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.
- 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
- Immerse the gel in 1× transfer buffer for 40 min.
- Activate the PVDF membrane with 99.5% methanol for 15 seconds.
- Immerse PVDF membrane, filter paper and sponge in 1× transfer buffer for 30 min before transfer.
- Complete a wet transfer at 500 mA, for 1h, at 4°C using 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.
Antibody staining
- 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 image using darkroom development techniques for chemiluminescence, or normal image scanning methods for colorimetric detection.