Photo courtesy of AJC1
Making your Western blocking solution – it is often a step that is done quickly just before you take your blot apart. You probably don’t give it much thought. But like all other steps at the laboratory bench, a little attention to detail goes a long way in improving your results.
Here are 5 quick tips that are sure to help you make the most of your blocking solution.
Make your Western blocking solution fresh
Proteins, detergents -the reagents in blocking solutions are perfect feeding grounds for microorganisms. If you let your Western blocking solution sit around for too long (even at 4°C) you risk the growth of bacteria and fungus which can destroy your Western blots.
Filter, filter, filter
Ever wonder what causes the random black constellations on your blot? These are usually caused by particulates – either from dirt or undissolved materials. An easy way to avoid this is to filter your Western blocking solution prior to use.
Try different blockers
It’s easy to keep using the same type of Western blocking solution for every experiment. But sometimes you need to mix things up (pun intended). Milk, BSA, single proteins and even protein-free blockers can all be tried when optimizing your Western.
Watch the salt
PBS or TBS – does it make a difference? Most times, no. However, if you are using alkaline phosphatase for detection, better stick with TBS as phosphate buffer can interfere with the enzyme.
Use detergent to clean it up
A detergent such as Tween-20 can be added to your Western blocking solution at very low concentrations to improve blocking (e.g. 0.05%).
So those are our quick tips to improving your blocking solution. Do you use any tricks when making your solutions?