Western Blotting Pt. 2

Hello everyone!

This week I wanted to wrap up my tips and tricks for Western blotting, specifically focusing on improving the antibody signal and detection. Without any further ado, let’s get into it! 

Set up a protein gel where each lane is subjected to different conditions: 

Although primary antibodies frequently come with suggested blocking solutions and antibody dilutions, you never know what condition will work best for you and your cell type. When setting up the initial gel, make sure that you load it so that most wells can be subjected different conditions, some of possible options I shall outline below! (Just don’t forget to run multiple protein ladders so you know where to look for your protein!)

Experiment with different protein concentrations: 

I’ve noticed that frequently, the main issue of me not being able to detect my protein of interest lies in its low abundance in my cells. It’s important to test a range of protein concentrations, ranging from 5-20μg, in all your samples of interest. This is because you don’t know initially what the ideal concentration of protein is, and whether your experimental conditions are altering its expression. 

Try blocking your membrane with both milk and BSA! 

On average, most people use 5% milk to block their membranes for one hour at room temperature. And although this typically works for most primary antibodies, sometimes it does not give a very clean signal. I’ve noticed that this is frequently the case with antibodies that were made in-house, rather than purchased commercially. BSA is your other membrane blocking option. The drawback is that this is the slightly more expensive reagent to use, but you can be sure that will always work for protein detection. 

Fiddle with concentrations of primary and secondary antibodies: 

Frequently, commercially bought primary and secondary antibodies will come with suggested concentrations. However, it’s always a good idea to try a range of concentrations to determine what will work best for you technique! Also don’t forget try out those different concentrations when diluted in both milk and BSA, as you won’t know what will give you a better signal until you try it! Secondary antibodies are frequently diluted at a much lower concentration, but if you have enough product, I’ve found it helpful to bump up the concertation a tad if you want to have a brighter signal. Just make sure to wash your membrane thoroughly before imaging! 

Consider using a primary antibody enhancer: 

I discovered these types of products this year and they have been revolutionary for those tricky proteins that are difficult to detect (or when you have a very minimal protein sample to work with). Antibody enhancers, such as Thermo Fishers Pierce™ Western Blot Signal Enhancer, increase the binding of the antibodies to the protein of interest, and as a result, amplify the signal when visualised. 

That’s all for this week- I hope these tips are helpful to you guys, and stay tuned next week for more tips and tricks!  

Product: ThemoFisher Western Blot Signal Enhancer (Catalogue number: 21050) https://www.thermofisher.com/order/catalog/product/21050#/21050