Western Blotting
Hello everyone! This week, I want to talk about Western blotting (part 1), a standard lab procedure used to detect the level of protein expression within a tissue or cell sample. Although standard, it can sometimes be a tricky process, so today I thought I’d highlight some of the tips and tricks I’ve learnt from my experiences.
Dealing with a low protein level sample:
There might be times after spinning down your protein sample you see that the pellet you have accumulated is rather small. If this is the case, I’d recommend resuspending it with a smaller volume of protein lysis buffer. Typically, the total volume that a protein well can hold is from 15-30μL (as an example, the volume of a well in Bio-Rad’s Protein Gradient Gel is 15μL). So if you are worried that you protein concentration might be too dilute, aim to have your protein lysate within these volumes.
However, sometimes you might accidentally resuspend your protein sample in a larger volume and realise later on that in order to get your desired protein concentration, you require a larger volume of protein lysate that the SDS-PAGE well can hold. If this is the case, use this clever trick I was introduced to in lab this year. Ensure that your SDS-PAGE gel has a a solid amount of stacking gel. Load the maximum amount of protein lysate that you can into your wells. Set the voltage to around 80V, press run, wait around 1 minute, and stop the current. This should have allowed your protein lysate to exit the well and into the gel, leaving the well free for you to load more protein lysate! After you have repeated this step for the amount of times needed (the maximum I’ve managed is 3), run your gel at a lower voltage than usual, this shall allow all the protein lysate to “catch up” to one another, and give you a clean strong band! Which brings me onto my other tip:
For a better gel, run your SDS PAGE at a lower voltage:
Although not necessary for lower weight proteins, if you are working with proteins of a larger kDa, I’d recommend running your gels at a lower voltage. This it ensures that the higher molecular weight proteins have a better chance at separating. Furthermore, it’ll make your protein samples are run evenly, and shall keep the temperature of your buffer in your tank low, which ensures that your proteins don’t “burn” (ending up in smiley faces).
Ice Ice Baby is KEY For Western Blot Transfer:
The thing I cannot recommend more is ensuring that the temperatures for your Western Blot wet transfer are as cold as possible. This can either be accomplished with; putting an icepack into the transfer back and completing the transfer in the cold room, burying the closed transfer pack in a huge box of ice, or cooling your methanol in the freezer aforehand. Once again we are trying to maintain the buffer temperature at a low, increasing the chances for a successful transfer!
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!
Mentioned materials:
Biorad 4-20% Gradient Gel, Catalogue Number: 4561096