j*****a
发帖数: 92 | 1 Amicon ultra-15 centrifugal filter (10 K) 浓缩38 K的蛋白,穿滤. 浓缩液，A3.5 (
280 nm)滤出液 A2.7 (280 nm).是质量不好? 以前也出现过，换一个就好了。这次试了
一盒filter都穿滤. | y******n
发帖数: 8667 | | a*****y
发帖数: 269 | 3 spead should not > 4000xg | o*****r
发帖数: 156 | 4 This should not happen.
You know that you should never trust the Abs value when it's more than 2.
1. What is your reference in UV measurement?
2. Do you have anything in the buffer that absorbs at 280nm, for example
imidazole?
3. What't the extension coefficient of your protein?
5 (
【在 j*****a 的大作中提到】
: Amicon ultra-15 centrifugal filter (10 K) 浓缩38 K的蛋白,穿滤. 浓缩液，A3.5 (
: 280 nm)滤出液 A2.7 (280 nm).是质量不好? 以前也出现过，换一个就好了。这次试了
: 一盒filter都穿滤.
| m**z
发帖数: 787 | 5 re this.What is the 280/260 ratio?
i had measured flow through from amicon before and it gave me high 280nm
reading. But when ran on a gel, there is nothing.
I think usually if you don't spin it with higher speed than you supposed to,
it should work just fine.
【在 o*****r 的大作中提到】
: This should not happen.
: You know that you should never trust the Abs value when it's more than 2.
: 1. What is your reference in UV measurement?
: 2. Do you have anything in the buffer that absorbs at 280nm, for example
: imidazole?
: 3. What't the extension coefficient of your protein?
:
: 5 (
| o*****r
发帖数: 156 | 6 Yes, spin at the recommended speed.
We use Amicon very often to concentrate proteins to ~10mg/ml.
I always put blank buffer in it and spin 10-15 mins, then spin my real
sample.
By doing this, the trace amount of glycerol (and other soluble stuff) on the
membrane get washed away.
And you do get a better UV measurement. My flow-thru was never higher than A
0.05 at 280 nm.
Occasionally, the protein might absorb to the membrane, but never ends in
the flow-thru more than 5%.
to,
【在 m**z 的大作中提到】
: re this.What is the 280/260 ratio?
: i had measured flow through from amicon before and it gave me high 280nm
: reading. But when ran on a gel, there is nothing.
: I think usually if you don't spin it with higher speed than you supposed to,
: it should work just fine.
| j****s
发帖数: 1366 | | s*******e
发帖数: 1010 | 8 what is in ur protein sample is most important. If it is hi-imidazole
elution, there will lots of small molecules have absorbance at 280nm and
they will go through membrane very easy |
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