发信人: rmchen (Raymond), 信区: Biology
标 题: Re: SOS! 请教如何防止蛋白质在sample buffer 中沉淀
发信站: The unknown SPACE (Sat Sep 21 08:15:40 2002) WWW-POST

Good point!

I am sure that the precipition was caused by potassium ions which complex with
SDS and become insoluble. You should remove potassium ions from your SDS
extraction solution by using sodium salt.

I do think your protein concentration is too high because I even used much
higher protein concentration for my protein 2d electrophoresis. But for
SDS-PAGE, you can not load too much protein even with a concentrated sample.
You still have to dilute your sample. I usually load 20-30 ug for my
self-made mini-gel.

If that was still a problem, you can try adding 8 M urea for keeping proteins
soluble. Not Guanadine HCl because this cause SDS precipition as well.

【 在 moyeveil (摩耶之幕) 的大作中提到: 】
: The concentration is too high for SDS-PAGE. I don't think one can get a good
: staining using such high protein concentration. But I don't think that's the
: cause of precipitation. The cause is potassium.
:
: You guys shall be very careful when you sample for SDS-PAGE contains
potassium
: for the solubility of potassium-SDS is very poor. Always get rid of
potassium
: first before adding SDS sample buffer.
:
: 【 在 sunnyday (飞鸟) 的大作中提到: 】
: : Yes, the concentration is high. And denature is also another factor.
: : You may try dilution or simply do a pre-spin at medium speed to seperate
: those
: : denatured proteins, since you don't really care about them in your assay.
: :
: : 【 在 golem (狗愣小岩) 的大作中提到: 】
: : : all your samples got precipitated?
: : : I think dilution is worth trying.
: : :
: : :
: : :
: : : 【 在 magnolia (麦田守望者) 的大作中提到: 】
: : : : 最近我在做chromatin binding assay. 这个方法是这样的：
: : : : 1.spheroplasting cells
: : : : 2.resuspend the spheroplasts in lysis buffer,add Triton X-100 (final
: : : : concentration is 1%) to break cells and release soluble proteins
: : : : 3.Whole cell extract is spinned at high speed to pellet Chromatin and
: : : : chromatin binding preoteins
: : : : 4. whole cell extract, supernatant after spin and resuspended
chromatin
: : pellet
: : : : are added by SDS-loading buffer and subjected to SDS-PAGE and then
: western
: : : : blot
: : : : 我发现蛋白质在加了sample buffer 之后，就会沉淀出来，boiling 也没有用。
请
: 问
: : 这是
: : : : 什么原因呢？我老板说可能蛋白质浓度太高了，浓度大概是8-9mg/ml，真的太高
了
: 吗
: : ？如
: : : : 何才能解决沉淀的问题呢？ 肯请有经验的人不吝赐教，在下感激不尽。/bow
: : : : components of lysis buffer:
: : : : 0.4M sorbital
: : : : 150mM potassium acetate
: : : : 2mM Magnesium acetate
: : : : 20mM Pipes/KOH PH 6.8
: : : : proteinase inhibitor: 1mM PMSF
: : : : 10 ug/ml Leupeptin
: : : : 1 ug/ml Pepstatin
: : : : 10 mM benzamidine HCl
: : : : TPCK
: : :


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