Getting your lysis buffer to work can sometimes be a science of its own. In this article, we point out nine issues to consider that could be throwing a wrench in your process, and we attempt to help you solve them. One of the biggest lessons when it comes to working with your cell lysis buffer is to not get caught up in trying to make it work. When you can find quick fixes, or cost-effective products that will reduce time spent, you should always consider using them.
1. Do you suspect it’s not working?
If this is your first attempt or you just have this feeling it’s not going right, this is a great tip. You can visualize lysis through a microscope, which will let you know early on if your buffer is working the way it’s supposed to. Other ways to monitor the release of proteins are by using a Bradford, Lowry or other assays. Typically, lysis buffers work pretty fast, so if you’re not seeing results right away, then it’s time to troubleshoot.
2. What is your detergent concentration?
A lot of times this can be your issue. Check the concentration of your detergent. When using a nonionic detergent, you want to make sure it’s in the 1.0% range. On that note, the ratio between detergent and membrane mass is important to lysis. Therefore, when the detergent amount is limiting, too many cells can take a toll.
3. Are you making your buffer?
This relates to number two. Preparing your own buffer can get tricky when it comes to determining the appropriate concentrations of other important reagents, especially if you're new at doing this. To simplify this process, it can be helpful to use kits that come with all the necessary reagents for. It might sound a little more expensive to go this route, but it saves you time determining how much Tris, EDTA and other products to use for your lysis buffer. Take a look at these GoldBio lysis buffers and buffer kits, which don’t cost much compared to other major brands, but they hold the same quality (if not, better). Remember, time is money, too, and optimization is key.
4. Are your proteins salt-resistant?
This is a fairly simple fix, but it might require some further experimentation. If your proteins are salt-resistant, then you may want to include an ionic detergent as well.
5. What kind of cells are you working with?
Something to keep in mind is the cells you’re working with and whether or not your treatment of these cells is actually compatible. For example, you may not simply be able to use a PBS wash for certain cell types, at least not PBS alone. In some cases, without using Ca2+ or Mg2+, you may end up with a low protein yield.
6. Are you optimizing your use of protease inhibitors or inhibitor cocktails?
This section has multiple parts because protease inhibitors are so important to the process.
a.First, let’s talk about storage – it is always important to use fresh protease inhibitors before you lyse your cells. If you store them in your lysis buffer, even at 4 °C, they will go bad after 20-24 hours. You can extend this if you store your protease inhibitors in buffer at -20 °C; that will buy you a few weeks. Ultimately, the point is to only store them in your lysis buffer if you absolutely have to, and take great care in doing so. Otherwise, always add them right before you lyse your cells.
b.Do you have a frost-free freezer? If so, -20 °C may not buy you quite as much time. This is because these types of freezers are usually programmed to have warm and cool cycles, which is how it prevents frost buildup. For most reagents, this is not a problem; however, it’s not a good idea to store enzymes in these types of freezers. Instead, you might have better luck with a StrataCooler or storing enzymes in a -80 °C frost-free freezer instead.
c.Some researchers like to make their own cocktails since other reagent supply companies can be quite costly. Instead of fussing with determining the right amounts when making your own, save yourself the trouble and check out this selection of GoldBio protease inhibitor cocktails. The quality and price is great, and there’s no headache. Also, remember to keep your extracts on ICE!
7. Is this a matter of protein concentration?
If so, you can do an acetone/trichloroacetic acid (TCA) precipitation. This is useful when you’re trying to concentrate your sample from cell lysates that have remaining contaminants. Be sure to use chilled acetone, as the protocol states, and keep it on ice during the procedure.
8. Does it appear like you’re getting more DNA rather than protein?
It may not be your lysis buffer; you may want to troubleshoot this situation. DNAse I is a good start. If this problem continually arises, use a cell scraper. You can also use sonication; however, there is debate about whether this is a good idea depending on your proteins. Bottom line, though, save yourself the trouble of trying to figure out what’s going on. Instead, spending a little more on lysis buffer kits will save you so much in the end!
9. Is your protein fairly insoluble?
We have actually addressed that in an earlier article. In this situation, you’ll see a pellet during purification. The solution proposed is to consider using GoldBio’s denaturing agents such as urea or GoldBio’s guanidine-HCl. Both products will help when it comes to proteins found in inclusion bodies. The earlier article also pointed out that by using both denaturing agents, you may recover up to 75% of your proteins.
While this doesn’t cover everything, especially very unique circumstances that arise when working with specific cells or systems, this does give you some general tips on how to troubleshoot lysis buffer issues. As we said before, one of the biggest takeaways from this article is to optimize your experiments whenever possible. Store things properly, and consider using products that help save you time. It’s always great to make it yourself, but when it doesn’t work, you end up losing much more than your sample and used reagents.
Share some of your tips so we can build a bigger list. And make sure you check out some of our other troubleshooting articles, which have been very helpful for our researchers.
Bursting the Bubble, Slaying the ‘Rat King’
Troubleshooting: Protein Expression
Troubleshooting: Purification of Tagged Proteins
GoldBio Marketing Coordinator
"To understand the universe is to understand math." My 8th grade
math teacher's quote meant nothing to me at the time. Then came
college, and the revelation that the adults in my past were right all
along. But since math feels less tangible, I fell for biology and have
found pure happiness behind my desk at GoldBio, learning, writing
and loving everything science.
Category Code: 88253 79103 79107