Our lab purified some soluble protein and kept them at -80, but the concentration of the protein varies when we evaluate them again. Some of them increased while some of them decreased. I wonder if the changes in concentrations resulted from protein solubility? And what factors could potentially affect it?
About what factor are we talking with the concentration changes? 10x? 5x? 1,1x?
If the change is only small, this might be due to measurement errors. What can happen in the freezer is that water evaporates and accumulates in the cap of your tube. When you de-freeze your tubes without a short centrifugation to collect all liquid in the bottom, its possible that you loose some water.
Sometimes a freeze/thaw cycle will cause proteins to crash out of solution or form aggregates- both can affect concentration measurements. Adding a cryo-protectant such as glycerol, sucrose, or DMSO can help. You can do a test, freezing small aliquots of your protein in a range of glycerol, sucrose or DMSO concentrations (say 0-20%), thawing the next day and measuring the concentration. This will show you what condition works best for your protein. Also, I always did a high-speed spin after thawing to remove any aggregates that may have formed before I used the protein. Another thought - do you flash freeze in liquid N2 or allow them to freeze slowly? For fragile proteins, flash-freezing may work better than simply placing them in the freezer. Having said all this, I had a protein that definitely behaved better when stored at 4degC. Sometimes you just have to try different conditions until you figure out what makes your protein happiest. Good luck!
Possible factor:
-Human mistakes in the meassurement process (after and before)
-The nanodrop is not well calibrated
-Precipitation
-Natural degradation processes.
Are you using protease inhibitor?
Anyway is virtually imposible not to lose protein during the process. It depends on the proteins you are working with. Make sure that you are using ice during the whole process and be accurate. The concentration may not decrease more than the -15% after all. You always have to meassure more than 3 times with the nanodrop and calculate the average.
Depends on what buffer your protein is stored in. Proteins stored in phosphate buffers have a tendency to precipitate and lose out on concentration. Tris buffers are preferred to store proteins at -80 deg C.
Also do you store the aliquots of the buffer that your protein was stored/dialysed in as you can use the same buffer stored in the same condition to baseline on spectrophotometer.
Nanodrop not ideal in my opinion for protein concentration measurements. Nanodrop for DNA is fine but not for proteins.
As Omar previously said protein may have indergone degradation hence the problem.
Stick to the same spectrophotometer for measurement as data from different instrument varies. Also, data could vary from the same instrument on different days but not so much.
It is best to store proteins with 10% glycerol in your buffer and flash freeze in liquid nitrogen before putting them in -80 deg as it prevents protein damage and keeps it stable.
All of the previous comments are clearly right, but another factor can also explain your problem : some proteins are quiet sticky and have tendency to adhere to sample tubes, thereby modifying the concentration of solutions. Using siliconized tubes can overcome (or reduce) this particular problem.
Besides all remarks st forward by others, the concentration range of your sample is very important because it dictates the method for protein determination. I wonder if you use Lowry or Bradford method because the sensitivity difference can result in error.
- Badges
- Science method
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Methods
- Protein Purification Techniques