So you’ve decided to take the plunge and go for a total stain to normalize your Western blot. With some beginner’s luck, it might turn out better than you have ever imagined…..or it might need some improvement.
Try these helpful tips to improve your total protein normalization results:
1. Optimize your conditions
Before you do the all-important experiment, take some time to practice with the stain or dye. Run a protein dilution series on a gel and go through the staining procedure. You will get some practice and some valuable information: you will determine the linear range of protein detectable by the dye/stain. Use that information when determining how much protein to load in a lane.
2. Mind the edge for total protein normalization
Dyes and stains can concentrate on the edges of your blots causing them to stain darker. Avoid the halo effect:
- Now is not the time to be skimpy with your reagents. Use enough stain or dye to completely cover your blot.
- Shake things up. Put your blot on a shaker or rocker while staining to distribute the dye/stain evenly. If using a rocker, rotate the blot 90 degrees halfway through the incubation.
3. Load lanes in duplicate or triplicate randomly across the gel
Let’s face it – sometimes things happen and a part of the gel becomes unusable or difficult to interpret. If you distribute the same sample in different places throughout the gel, you will increase your chances of obtaining the “perfect” data. And hey, if they all look good, you will have lots of usable lanes to incorporate into your data.
4. Use good practices when preparing your samples and loading your gel
Attention to detail can go a long way in improving your results.
- Make sure you determine the total protein concentration of each sample and load the same amount of protein per lane.
- Recover all of your sample before you load. Centrifuge sample tubes after heating and prior to loading to make sure all of the sample is collected on the bottom of the tube.
Photo courtesy of Scott