I am using NP-40 lysis buffer to extract membrane proteins. Last two attempts I did not get these proteins. How can I use this detergent containing buffer to extract membrane proteins. Thanks
You can use Triton X-100 or Tween 20 or Tween 80 in concentration range of 0.1-0.5 W/V of lysis buffer(Tris.HCl + NaCl , pH8.0-9.0). Incubate for some time and do lysis in mild condition to extract the POI. you can give Deoxycholic acid(DOC) wash to extract POI depending on its Concentration. After Extraction you can add 2 M Urea just to stiff your protein in proper conformation which may not interfere in ELISA as well.
Rupen had some very good suggestions and leading you toward a RIPA Buffer. You might entertain a RIPA Buffer used for Immuno-precipitations - they are usually cocktails of those detergents he listed. Do you have an antibody - that might help you on the backside of purification after extraction by using Immuno-precipitation. Then you can prove extraction and purification by western blot end point - 2 steps to demonstrate proof of concept.
Next would be to scale up an extraction/purification scheme.
Another detergents you might want to consider is Empigen BB which is a dodecyl-derivative of Glycine. That in a cocktail with Sodium Deoxycholate and proteinase inhibitors might help you out. Try to keep it simple as possible and follow the foot steps of those that have proven track record of pulling off what you're attempting.
I am in industry and have been a protein reagent developer.
Good Luck - Look forward to hearing of your progress.
Thanks Tao, Bhavsar, Weaver. Certainly, detergent combination will work better to extract membrane proteins. I think any ionic detergent would be considered for membrane protein extraction. Would you please explain me how these detergents are employed to extract proteins?