Seven tips to improving your Western Blots:
· 1. Uniformly distributed high background?
o You may have insufficiently washed. Increase the number of washes and buffer volume.
§ Make washing easier with
Blot Boxes.
o 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?
o 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
o 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.
o 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?
o 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?
o Try the deeper wells from Expedeon! Easily hold 35ul of sample without bleeding over to another well!
o Try gel-loading tips. Available in flat or round, they make gel loading easier!
· 6. Worried about pre-cast gels expiring or losing resolution before you can use them?
o 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!