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TCA precipitation from High% sucrose solutions - (May/05/2010 )

I am attempting to precipitate protein from fractions of a sucrose gradient. The most concentrated samples are 55% w/w (about 68% w/v) sucrose and have a volume of 1 mL in NTE (150mM NaCL, 20mM Tris, 1mM EDTA).
My technique has been:
-Add TCA to 14%.
-Rock O/N at 4C.
-30 min. 22,000g, decant.
-2 washes with -20C 100% Ethanol (same spin as above between)
-Vac spin dry.

-Suspend in Sample buffer with 0.5M urea and 0.2M tris (8.8) (also BME, DTT, SDS)
-RT O/N.
-Full power sonication 5 minutes.
-Boil 15 minutes.
-Load, run...etc.

In this process I seem to be losing about 50-75% of the input based on control experiments. Is there something I can change to increase the efficiency?

My other concern is that I absolutely MUST be getting equal efficiency of precipitation at the whole range of sucrose concentrations (2.5 - 55%).
Does anyone think that the high sucrose concentration would cause a problem... and how can I overcome it?
I HAVE noticed that BSA in particular seems to precipitate with less efficiency at higher sucrose concentrations... but some other proteins don't appear to be affected.
Input? Recommendations? Freelance verse?

-Another Jake-

I ran sucrose gradient of 5-35% (w/w) and proceed with TCA precipitation. I am not sure the low efficiency is caused by the high concentration of the sucrose, b/c I have the same issue with my system. People say that the major problem with TCA precipitation is to get the precipitate to dissolve. I looked at your protocol and am not sure about is the RT O/N after re-suspension. Is it too long? I saw people do 50C for 1h. I don't know if it helps. I am trying that myself. Please let me know if you find a better way. Good luck! :)

Another Jake on May 5 2010, 11:25 AM said:

I am attempting to precipitate protein from fractions of a sucrose gradient. The most concentrated samples are 55% w/w (about 68% w/v) sucrose and have a volume of 1 mL in NTE (150mM NaCL, 20mM Tris, 1mM EDTA).
My technique has been:
-Add TCA to 14%.
-Rock O/N at 4C.
-30 min. 22,000g, decant.
-2 washes with -20C 100% Ethanol (same spin as above between)
-Vac spin dry.

-Suspend in Sample buffer with 0.5M urea and 0.2M tris (8.8) (also BME, DTT, SDS)
-RT O/N.
-Full power sonication 5 minutes.
-Boil 15 minutes.
-Load, run...etc.

In this process I seem to be losing about 50-75% of the input based on control experiments. Is there something I can change to increase the efficiency?

My other concern is that I absolutely MUST be getting equal efficiency of precipitation at the whole range of sucrose concentrations (2.5 - 55%).
Does anyone think that the high sucrose concentration would cause a problem... and how can I overcome it?
I HAVE noticed that BSA in particular seems to precipitate with less efficiency at higher sucrose concentrations... but some other proteins don't appear to be affected.
Input? Recommendations? Freelance verse?

-daxiaopenny-