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Multiple reactions buffer and enzymatic special activity

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Multiple reactions buffer and enzymatic special activity

Postby dalin » Wed Oct 17, 2012 3:40 am

In the scientific papers, If get stuck with the way they write that is kind of arbitrary for instance if you have an assay Buffer (10X) that is concentrated and if I dilute 3 ml of that by adding 30 ml of water which will bring the buffer concentration to 25 mM Tris-HCl, Ph 8.0, containing 140 mM NaCl, 3 mM KCl, and 1 mM MgCl2 and 1 mg/ml bovine serum albumin?
Can you please explain in details???

Also with the enzymatic reaction I gut stuck with information given for the enzyme for instance I have this assignment: an enzyme has is provided in a concentration of mg/ml, activity mU/ml and specific activity of pmol/min/mg that is the one unit required to give pmol of product per minute in buffer condition containing 40 uM of substrate and 100 uM of co-substrate. Can you please help to understand this? And how this is relate to the design of the conditions for the enzymatic assay.
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Postby dalin » Wed Oct 17, 2012 3:45 am

sorry, i mean U/ml not mU/ml , also U/ug and one unit = 1 pmol/min those refer to how enzyme activity is defined and are important for the design of the conditions and concentrations of enzyme/buffer/substrate/co-substrate. Please help me?
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Postby JackBean » Fri Oct 19, 2012 3:09 pm

I'm sorry, but I don't understand your questions.
http://www.biolib.cz/en/main/

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Postby dalin » Sun Oct 21, 2012 5:29 am

In the scientific papers or in protocol procedure, sometime do not understand the way they write their experimental data. For instance, a Buffer that consists of a multible reagents and labeled (10X). I was asked to dilute it by taking 3 ml of that 10x buffer and add 30 ml of distelled water and I was told that this will bring the buffer concentration 1X
With final concentrations of 25 mM Tris-HCl, Ph 8.0, containing 150 mM NaCl, 3 mM KCl, and 1 mM MgCl2 and 2 mg/ml bovine serum albumin?
Can you please explain in details??? How the buffer was made in detail initially and then how it was diluted from 10X to 1X.
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Postby JackBean » Sun Oct 21, 2012 11:07 am

To make it 1x you need to dilute it 10-times, thus for 3 ml you need to add 27 ml, not 30. Or better fill it up to 30 ml. That's a difference.
The way to prepare such buffer is simply prepare buffer with 10-times higher concentrations, i.e. 250 mM Tris, pH 8.0, 1.5 M NaCl, 30 mM KCl, 10 mM MgCl2 and 20 mg/ml BSA.
http://www.biolib.cz/en/main/

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Re: Multiple reactions buffer and enzymatic special activity

Postby dalin » Sun Oct 21, 2012 11:18 am

Thanks for this explanation, but I still need your help! How to prepare multible reagent buffer how do I calculate and prepare the concentrated buffer that is 10-times higher concentrations of 250 mM Tris, pH 8.0, 1.5 M NaCl, 30 mM KCl, 10 mM MgCl2 and 20 mg/ml BSA. Let's say my final volume is 500 ml. It is very confusing for me.
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Postby JackBean » Sun Oct 21, 2012 11:39 am

The usual way just as for 1x buffer by m = c.V.M (i.e. 250 mM . 500 ml . 121.1 g/mol for Tris)
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