I used RIPA buffer during protein extraction which I found it interferes with subsequent ELISA possibly due to SDS in the buffer. Is there any way to recover the protein from it?
RIPA buffer does not need to have SDS in it. There are many RIPA formulations now, but older formulations did not have any SDS, just NP-40. Some people add deoxycholate. You should try it without SDS and see how it works. Dialysis will not remove SDS. It sticks to the protein. Detergents are generally hard to dialyze because they form micelles that are too big to go through the membrane. SDS will form mixed micelles with the other detergent(s) in your formulation. Acetone will get rid of the detergents, but may also denature your protein and/or render it insoluble. Since it is impossible to predict this, you would have to try it out to see if it works for you.
Hi Fadi,
I would rather go for a protein precipitation with acetone, that would clean your samples from the SDS. Here is a link to a protocol:
https://tools.thermofisher.com/content/sfs/brochures/TR0049-Acetone-precipitation.pdf
Hi
If you have small volume of sample, you can concentrate your sample using centricon (Millipore, 3KDa) that will remove buffer from your sample and increase the protein concentration.
Simply tou can use protein extraction kit.It binds proteins to a disposible column and you can elute it with another buffer.
You could perhaps do an old-fashioned dialysis. Using the little dialysis devices is best, but if your budget is limited you can use tubing with clamps. Protocols can be found in the good molecular biology manuals.
RIPA buffer does not need to have SDS in it. There are many RIPA formulations now, but older formulations did not have any SDS, just NP-40. Some people add deoxycholate. You should try it without SDS and see how it works. Dialysis will not remove SDS. It sticks to the protein. Detergents are generally hard to dialyze because they form micelles that are too big to go through the membrane. SDS will form mixed micelles with the other detergent(s) in your formulation. Acetone will get rid of the detergents, but may also denature your protein and/or render it insoluble. Since it is impossible to predict this, you would have to try it out to see if it works for you.
Ignore my suggestion given what Norbert said. I hadn't known this.
Common RIPA components
PBS : Salt prevents non-specific protein aggregation
Tris-HCl : Buffering agent prevents protein denaturation
NaCl : Buffering agent prevents protein denaturation
1% Nonidet P-40 or Igepal CA-630 : Non-ionic detergent to extract proteins, form lipid micelles
1% Triton X-100 : Non-ionic detergent to extract proteins, form lipid micelles - to use in place of Nonidet/Igepal
0.5% sodium deoxycholate : Ionic detergent to extract membrane protein and isolate lipids
0.1% SDS : Ionic detergent to extract membrane protein and isolate lipids
PMSF : Stock Solution 100 mM (100x stock) or 200 mM in Isopropanol; use @ 1 mM. Store PMSF solution up to 6 months @ 4C.
EGTA : Protease Inhibitor, Prevents protein degradation. You can make your own, or several vendors have convenient crushable pills that form a protease inhibitor cocktail solution.
Na3VO4 (Sodium Orthovanadate) : Tyrosine phosphatase Inhibitor; hydrostatic interference of active sights of phosphatases
NaF (Sodium Fluoride) : Serine/Threonine phosphatase Inhibitor; hydrostatic interference of active sights of phosphatase
Phosphatase Inhibitors
Phosphorylation/dephosphorylation of proteins influences hydrostatic relationships. Proteins undergo covalent attachment of a phosphoryl group (phosphorylation) at serine, threonine, or tyrosine residues. Phosphate groups are removable via protein phosphatases. During the extraction of phosphorylated proteins from cell and tissue, there are advantages to preserving the phosphorylation states of total protein.
(-)-p-Bromotetramisole oxalate
Cantharidin
Calyculin A
Discodermia calyx
Imidazole
Sodium Fluoride
Sodium Molybdate
Sodium Orthovanadate
Sodium Tartrate Dihydrate
Thank you all for your contribution, it is highly appreciated.
Actually, I ll try Amicon Ultra-0.5 mL Centrifugal Filters to wash the protein from RIPA buffer and reconstitute it with the compatible buffer for ELISA.
As others have said, dialysis will not remove the detergents. You can try binding it to an ion exchange small spin column from Thermoscientific, wash and then elute. This will ensure your protein does not get denatured if you need it that way for ELISA determination. Although if SDS is present in the RIPA buffer, it may already be unfolded.
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