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Improving your Western Blots
Seven tips to improving your Western Blots:
1. Uniformly distributed high background?
- You may have insufficiently washed. Increase the number of washes and buffer volume.
- Make washing easier with Blot Boxes.
- Antibody concentrations too high
- Make sure to optimize your antibody concentrations by running strips at varying concentrations to determine best concentration for your samples.
2. Weak or no signal?
- Your protein may have not transferred well.
- Check your transfer buffer make-up and blotting procedure. Stain gel to visualize proteins remaining in gel.
3. The Ladder Lane is black
- The antibody may be reacting with your MW (ladder) marker.
- Add an empty lane between the MW marker and sample lane.
4. The band of interest is too low/too high on the blot.
- Separation is not efficient.
- Change the gel percentage: A higher percentage for small protein, a lower percentage for larger proteins. Or try a gradient gel.
5. Ripped resolving gel when moving to the cassette for transferring?
- Try Run Blue™ pre-cast gels from MIDSCI. They are stronger and resist tearing!
- No more piecing together torn gels!
6. Sample bleeding over to another well or difficulty loading?
- Try the deeper wells from Expedeon! Easily hold 35ul of sample without bleeding over to another well!
- Try gel-loading tips. Available in flat or round, they make gel loading easier!
7. Worried about pre-cast gels expiring or losing resolution before you can use them?
- Expedeon Run Blue™ gels have a 2 year shelf life with no loss in resolution. Use them confidently from purchase date to 2 years later!
