I am planning to use RIPA lysis buffer on cell debris to study membrane-bound protein fraction. Yet, I found that mere vortex of the cell debris in RIPA buffer is not sufficient to release proteins from the cell debris. Therefore, I would like to ask for suggestions. Does the process also require specific treatment such as heating of the sample to a certain degree or freezing, etc?
you mean that you lise your cell in some buffer and then you pellet the cell lysate. On the pellet you have your cell debris and this you are trying to analyze in RIPA? Am I understand well? So, in this case, you can try to incubate your membrane fraction 20 min on ice with occasional vortexing, then spin again (top speed, 10min@4°C) and subsequently try to dissolve your pellet with a more strong lysis buffer (I.e. 100mM NaCl, 50mMTris pH 7.4 and 1%SDS). With this, all the protein will (hopefully) be released
Generally speaking, you need to sonicate membranes (eg 3 x 10 sec) in order to completely suspend them. Don't forget to cool the sample on ice in between sonications.
it is better to use sonicator than freezing and thawing. you need to add strong lysis buffer during sonication. and also maintain cool temperature during sonication.
in this case it is better to use SDS in lysis buffer
I have used RIPA a lot and it is so strong and even lyze out the nuclear proteins. The trick is to let RIPA sit with your cell on ice for 10-20 minutes then either sonicate (15% output 10 sec X3) or use syringe (if the volume is really small). Even though we all call it RIPA but I do know several different versions of the cocktail by goggle it. SDS or triton X-100 are strong detergent and can help your extraction. One problem will be that RIPA is so strong so most of the time you will end up with some very sticky (DNA) jello at the bottom of the lysate that needs extract care to handle. I also experience a lot of wedge formation during electrophoresis using RIPA that is very annoying.
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