I prepared cell lysates for western blotting using RIPA buffer. After successive freeze thawing cycles, I can see precipitates and protein concentration went very low. Are there any methods to get the proteins back into solution?
I always thaw them on ice in order to prevent protease activity. In case I heat them to 37 C, I believe the protease will be active and I will get smears on the blot.
You definitely need to aliquot your lysates to avoid too many freeze-thaw cycles. Do you use any protease inhibitors? Is your only goal to run a western blot and identify the presence of your protein of interest? Is your PAGE gel under reducing or non-reducing conditions? You can denature your sample with 4M-8M Urea (pH properly) and your protein should go back into solution. Depending on the volume of your lysates, you can also filter (or pellet) out what has precipitated and reduce the volume back down (increase concentration) with a spin concentrator. Obviously your protein profile in the sample will be skewed after this. Best bet is to avoid multiple freeze thaws, even if it means making 50X 2 uL aliquots. Also, try not to use a lysate that has too high of a protein concentration (oligomers tend to form). You can do a western blot based dilutional analysis of your sample and see the minimum cutoff concentration needed to give you a positive result. Then dilute the sample accordingly and standardize the assay.
Many thanks James Coleman!
Yes it is better to avoid freeze thaw cycles but it's for next time, Lessons learned!
The samples are only for western blotting.
Yes there is protease inhibitor cocktail (HALT) including EDTA. The volume of my samples is approximately 100ul.
I can run western and find cutoff concentration but the problem is that the samples I am trying to compare with are fine and precipitates are only in certain crucial samples.
Any suggestions???
What Adam said. If all you want to do is SDS-PAGE, then you're in luck. It doesn't matter if your protein is precipitated. Boiling in reducing SDS buffer denatures and solubilizes every protein. The end-result is the same, whether your protein started out properly folded or mis-folded. That's kind of the point :)
Why do you use Ripa buffer for western blot? Use SDS-PAGE loading buffer (25mM Tris-HCl ph 6.8, 3%SDS, 25% beta-Mercaptoethanol, 50% glycerol) you do not even need to use proteases inhibitors.
Hi Paola,
Many thanks,
Yes we use RIPA buffer for western blots. And we keep the lysate in -20 till we load it on the gel and to load yes we use the loading buffer but we use LDS loading buffer.
Now, are you suggesting we make the lysis in this LDS buffer itself? and LDS will denature and inhibit the action of proteases itself?
Regards,
yes Motasim Masood, for western blotting lyse your cells in opportune quantities of SDS-loading buffer and boil for 5 min (or 95°C)
I have also noticed that the presence of NP40, triton-x100 or detergent different from SDS modify migration of proteins in SDS-PAGE, showing a different molecular mass
In that case Paola Di Bonito, do you not add any dye to monitor the gel running? because if you add something to the buffer for that purpose it is impossible to quantify the proteins.
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