Taming your Western

During our projects we’ve learnt a multitude of molecular techniques, but the most elusive so far has been mastering the Western blot. Before Christmas we’d completed our promoter trap constructions, using molecular cloning to transfer our genes of interest (Kinesin13, Ark2, Rap9 and Smc3) under the control of the AMA1 promoter to restrict expression. We're currently waiting for our malaria transfections to show any sign of success, and for the meantime working on Western blotting and fluorescence imaging of our GFP-tagged proteins. On Ching has previously posted a summary of her project, so to avoid repeating ourselves I'd like to share one of the challenges of our project: the Western blot.  

The humble Western blot has many uses throughout biomedical research, including disease diagnosis, antibiotic efficacy studies, and the FIFA 2014 World Cup doping tests. For our purposes, we'd like to demonstrate that the fluorescence shown on our images indicates our protein of interest. Jack will shortly be writing a post about our experiences with microscopy, and I'd like to talk through the many mistakes we've made with Western blotting.

Image courtesy of David Taylor

In striving for the good blot, our initial results all too often resembled the bad and the ugly, and frankly things didn’t improve until we altered several steps of our lab protocol. We hope sharing our experiences troubleshooting Western Blotting will save some frustration for other students, and at the very least provide a snapshot of numerous ways it can go wrong. For any newcomers to Western Blotting - or anyone who just feels like punishing themselves -  we’d recommend this starter paper as a background to protein blotting.

1) Protein Handling – Lysis, Denaturation and Loading

The ugly: poor lysis, degradation
and overloading. 

Shown is one of our first gels, and to this day I still remember the look on our PIs face. The underlying nature of a Western relies on the quality of the protein that’ll be used. Our projects have involved isolating parasite proteins from infected mice blood, using CF11 columns for leukocyte depletion and then lysing cells to gain access to our proteins. During our first few westerns, we were having difficulty with protein degradation during cell lysis, which was compounded by chronic overloading of protein onto the gel. After several smeared and blotchy attempts, we rewrote the lab protocols for the lysis steps, lowering temperatures by lysing on ice and centrifuging at 4^oC, incorporating protease inhibitors and also restricting repeated freeze-thaw cycles.

Throughout this process we’ve also learnt to select detergents depending on our protein’s hypothesised cellular location – NP40 may be fine for cytoplasmic Kinesin 13, but for the nuclear Ark2 Sarkosyl would have been a better choice. A general rule is that the more compartments between you and your protein, the harsher the detergent needed. 

2) Supervising your Western

The overheated: a cautionary tale 

Borrowing the words of the Upturned Microscope: ”Your western blot hates you. Never leave it unsupervised”. After setting gels running, it’s usual to notice rising bubbles alongside fluctuating voltage and amp values. We’ve also found it’s a good idea to keep half an eye on our westerns, checking at least halfway through the cycle and not leaving it unsupervised for too long. During one of our earlier gels, there was an incident with overused electrophoresis buffer that resulted in the gel just melting within the chamber. A cursory glance over the tank will quickly reveal if everything is running as normal: checking the dye front is symmetrical, the tank feels cool to the touch and your protein hasn’t run off the gel edge will save a headache later.

The disturbed: temperature and
electrolyte fluctuations

If running for an extended period of time, it’s also best not to disturb the tank. For one of our experiments we were attempting to resolve a high molecular weight protein which refused to leave its well. We were running two gels, the first for 1hr 30min and the second for 2hrs, to reach better resolution. We made a rookie error: running the two in the same tank. Removing the first gel at the end of it's run meant turning off electrophoresis, removing the gel and refilling the electrolyte solution - all resulting in salt redistribution and temperature changes. The difference between the ladder on our two gels can clearly be seen – one resembles bands and the other blotchy smears.

3) Transfer

We’ve found that thoroughly removing any trapped air from the sandwich is an essential step once it’s been assembled onto the transfer system. Murphy ’s Law states that any bubbles occurring will appear just at the right height to obscure your protein band, and if you’re really unlucky these bubbles can span several lanes. Remembering to equilibrate sandwich components in transfer buffer for 2 – 10 minutes before assembling and then rolling out any air will help. This equilibration period also gives the gel time to cool which avoids transfer problems, and most buffers contain methanol to reduce air retention within your sandwich.

4) The Antibodies

Have you checked your primary antibody species matches your secondary? If you’ve ever had a western with no signal, this could be the culprit. Whilst this may seem like an obtuse mistake, it’s particularly easy to make when using Western Reagent kits. Our lab uses WesternBreeze by ThermoFisherScientific, which contains two delightfully similar bottles of mouse and rabbit antibodies, and once produced a confusingly blank blot. It is also possible to strip membranes and begin again from the beginning, but as this isn't something we've tried yet we'd love to hear our viewers comments on the process.

5) Background

The obscured: high background.

After our initial problems with high background on our westerns, we increased the frequency of our washing steps.If there’s too much background on the western, we’veincreasing washing steps will help to resolve the issue. However, if the background is disguising smearing or low signal to background levels, you may wish to examine how you’re handling the membrane and check your antibody dilutions. If assembling your sandwich involves the membrane sliding over the gel repeatedly (or when developing has frequent repositioning of your film within the cassette) it’s also possible to create smears and blotches.

If you’re having similar troubles with your western, we’d love to hear about it! You can share your worst westerns in the comments. For any students looking for a more comprehensive troubleshooting gallery, we’ve found the SDS-PAGE Hall of Shame curated by Rice University to be particularly inspiring. Join us again next fortnight when Jack shares his experiences with fluorescence microscopy in our next post: “Fluorescence and Failure”. 

References

1.  Baume N, Jan N, Emery C, et al (2015) 
   Antidoping programme and biological monitoring before and during the 2014 FIFA World Cup Brazil. British Journal of Sports Medicine. 49: 614-622
   
   
2. Kurien, B. T., & Hal Scofield, R. (2015). Western blotting. Methods. 38(4): 283–293 https://doi.org/10.1007/978-1-4939-2694-7
   

   
   
3.  Johnson, M. (2013). Detergents: Triton X-100, Tween-20, and More. Materials and Methods, [online] 3 (163). Available at: www.labome.com/method/Detergents-Triton-X-100-Tween-20-and-More.html.