[Bookie]

Hello, I would like someone to explain me something. Suppose that we have 2 test tubes wich contain the following:

Test tube No1: 1ml lysozyme (6mg/ml Tris/HCl pH 8,0), 3ml 3,6M NH₂SO₄ and 2ml water
Test tube No2: 1ml lysozyme (6mg/ml Tris/HCl pH 8,0), 4ml 3,6M NH₂SO₄ and 1ml water

After centrifugation we measure the absorbance of these 2 test tubes and we find out that A for both of the test tubes is 1,653. How can this happen if we did the experiment by the book ? Is there any logic explanation behind it  and some scientific literature that explains it ? 

Thanks in advance

[Arkcon]

Does the book say for certain something else would happen?  If so, then each of your statements of fact, or descriptions of procedures are suspect, and you should recheck your notes.

Is there any logic explanation behind it  and some scientific literature that explains it ? 

I'd start with mys suggestion above, first.

[Bookie]

No, it doesn't say anything about it. I did ask my professor's opinion and he told me that it can happen and that he would accept the result of the experiment only if i have an adequate explanation for it and that's the reason i ask for your help.

[Yggdrasil]

Can you give more details about how you prepared these solutions?  Way in which the components were mixed is important for ammonium sulfate precipitations.

[Bookie]

With the following order
Lysozyme
NH₂SO₄
Water

[Babcock_Hall]

At what wavelength were the absorbance measurements made?  I am guessing 280 nm, but I would like to be certain.

[Bookie]

280 nm

[Yggdrasil]

Usually, when performing an ammonium sulfate precipitation, one takes precaution to ensure that the protein is not exposed to concentrations of ammonium sulfate higher than the final desired concentration (for example, adding the ammonium sulfate dropwise while mixing the protein solution).  Is this the case with your experiment (especially considering your order of addition)?

Do your absorbance values indicate that more lysozyme was precipitated than expected or less?

[Bookie]

First of all i have a mistake on the above values

Test tube No1 has the following ingredients

1ml lysozyme (6mg/ml Tris/HCl pH 8,0), 4ml 3,6M NH2SO4 and 1ml water

Test tube No2: 1ml lysozyme (6mg/ml Tris/HCl pH 8,0), 5ml 3,6M NH2SO4 , NO water

A=1,653

Actually i cannot answer to your question because i don't know the answer

[Babcock_Hall]

The molar absorptivity of lysozyme is 37,800 M^-1 cm^-1.  I suggest calculating the concentration of lysozyme using this information.

[Yggdrasil]

Wait, are you adding ammonium sulfate (NH₄)₂SO₄ or NH₂SO₄?

[Arkcon]

Bookie:, first of all, I'd like you to start by reading the Forum Rules{click}.  Briefly, we don't dump complete answers on these forums.  So you're going to have to come up with an answer on your own, we're going to try to help if the answer seems to be going the wrong way, or if you need confirmation of an idea you've come up with, but you seem to want a "literature" source for an undergraduate experiment.  You should try to realize, this is unlikely to exist.  I also gave you my "logic", I think you did it wrong, and you have to read your notes carefully to tell us/yourself where.  But one word answers per post will make this a very long thread that goes nowhere.

You've tried two concentrations on the same sample, and got the same result.  Why can't you say that?  Do you know for sure that one of them was supposed to work?  If you're sure, then yes you can't say that this was the expected outcome.  But if you're not sure, how will you know if this was supposed to happen, of if you did it wrong?  Or your instrument was broken, or you used the instrument wrong?

P.S.  Its a little odd that you got the same number, 1,653, each time.  Not 1653 and 1650?  Something like that?  That''s what I get from an HPLC when I run it, as an example.

[Bookie]

I was adding ammonium sulfate.

Arkcon i do understand the forum rules and i totally agree with them. At first, when i found out that 2 different samples had the exact A seemed to me a little odd as you 've mentioned already and i went to my professor and told him about the results. He told me that it can happen due to ammonium sulfate and he also mentioned that he wouldn't accept the result of my experiment as correct, unless i have an adequate explanation for it (probably he is interested to know whether i understand why my samples had the same A value). I am 100% sure that i did follow the instructions with the correct order. One other thing that i have to admit is that my biochemistry background is not good enough to explain him the reason.

I am not saying that my result is wrong but i don't have the scientific background to support it and i was wondering if a biochemistry scientist from the forum would be able to help me support my result. 

[Babcock_Hall]

Bookie,

Except for the point that Arkcon raised, your results do not seem entirely surprising to me.  Can you advance a reason why you think the absorbance values might be the same?  I don't think you have put forth a hypothesis yet.  Once you do that, we can help you test your hypothesis against your evidence.

[Babcock_Hall]

When you centrifuged the samples, was the liquid supernatant clear or cloudy?  What solid material did you see, if any?