I scraped the cells out of the flasks and homogenized them with mortar and pestle type homogenizer. I solubilized the membrane particles with 1% Triton X100. In order to separate the solubilized membrane proteins, I have to ultracentrifuge the samples. I need the ultra-centifugation to be in a fixed angle rotor. The volume of my sample is 8mL. What is the recommended centrifugal force (g), time and temperature? PLEASE do advise me on the recommended types of rotor, TUBES that can withstand extreme conditions like 100000g for 1 hour.
After adding the detergent you just need to separate debris
from the solubiized proteins. This will work in table top
centrifuge at 20 000g.
If you really want to go for the ultra centrifuge:
Polycarbonate tubes and Ti 70 rotor will do (45-55Krpm for 40-90min to get the liposomes pelleted),
but you might need to dilute your sample since half-empty tubes (25 cc) are less
robust. Ideal would be to use smaller scale ultracentrifuge (MLA ? - don't remember
exactly).
Thank you Misha !
Earlier, I tried 20000rpm (rotor JA20.1 - BC-Avanti-JE) for 2 hours with a polystyrene tubes and they were shattered. I now made a try, 100000g for 1 hour (BC-Optima XE100 - ultra centrifuge, rotor - SW-Ti 40) in a swing out type. Yet to quantify the membrane proteins. I will keep you posted about that.
Meanwhile, can I ask you for the optimal concentration of detergents (in my case, its Triton X100) to soulubilize and the incubation time? What are the factors associated with the proteins that determine the type of detergents to solubilize them?
Choice of the detergent is totally empirical, it is a book-long subject.
Triton is among less friendly since it interferes with 280 nm OD reading
and it is very hard to get rid off.
1 % concentration that is normally used for the solubilization and
1hr should be enough (you should see that solution gets clear).
About the centrifugation - after adding the detergent, most of the insoluble
fraction are protein aggregates. These are very dense and require very
little effort to be pelleted. Try table top centrifuge (cooled one) and use
eppendorfs, it will save you a lot of trouble.
A warning concerning Triton X-100: it is one of those detergents that form peroxides when stored in air. These can damage your protein. If you are going to use it for protein isolation, I strongly recommend using detergent that has been stored under inert gas. Get a fresh bottle, make a 10% solution, and store aliquots in the freezer. You can also buy 10% Triton X-100 sealed in ampoules from Thermo-Fisher (Surfact-Amps-100).
For ultracentrifugation post-solubilisation, given the volumes you are talking about, I would suggest the Beckman 90 Ti rotor (https://www.beckmancoulter.com/wsrportal/page/itemDetails?itemNumber=355530#2/10//0/25/1/0/asc/2/355530///0/1//0/) and 10.4 ml polycarbonate tubes (https://www.beckmancoulter.com/wsrportal/page/itemDetails?itemNumber=355603#2/10//0/25/1/0/asc/2/355603///0/1//0/). Fill these to the top, but don't spin at the top speed of 90,000 rpm - this is equivalent to almost 700,000 g. Instead, just spin at 100-150,000 g for 30-60 min.
I would strongly suggest that you do use an ultracentrifugation step and not a lower speed spin at e.g. 20,000 g. A lower force like this will never efficiently pellet partially solubilised protein aggregates, etc. You will see a clear difference in pellet size between spinning at 20,000 g and 100,000 g.
If you don't have access to a 90 Ti, and only a 70 Ti or 45 Ti, then you could easily dilute your samples post-solubilisation and use bigger tubes, keeping the detergent concentration above CMC.
One final point/question - why are you using Triton X-100? It's a pretty crude detergent and as mentioned can form peroxides. Probably the most popular detergent for solubilising integral membrane proteins is dodecylmaltoside (DDM). Unless you have a good reason otherwise, DDM is a good place to start. Even better - screen a series of detergents for solubilisation efficiency and protein activity.
As to the concentration of detergent to use, many people tend to just use 1% without optimising. One of the best ways to optimise this step is to prepare a resuspended membrane solution, determine its protein concentration with e.g. Bradford assay, and then prepare a series of dilutions e.g. from 5-0.5 mg/ml protein. Then solubilise all with 1% detergent (for 60-120 min at 4C), spin, and see which one gives the best solubilisation.
Hope that helps.
I agree with the excellent suggestions from Roger Bradley Dodd. However, you may want to start with a low speed centrifugation (10,000 g) to remove unbroken cells and cellular debris. Then you can solubilise the supernatant (i routinely use 1% DDM for 1H at 4C) and ultracentrifuge at 100,000 g or above for 1H at 4C. As Roger said, you can test a variety of detergent at 1% and later optimize its concentration (lower than 1% may well work too and save you money). Lastly to measure membrane protein concentration, i first precipitate the proteins with DOC and TCA, then do a Lowry.
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