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?
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 (
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%.
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【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.
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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