protein concentration - (Mar/13/2006 )

the concentration of a protein sample before and after being subjected to temperature induced stress conditions are different. the concentration of the stress induced sample is lower than the control sample. why is it so?
Concentration was measured spectrophotometrically at 280 nm as well as by lowry's estimation.

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some of the protein may have denatured and come out of solution.

also, did you mix after defrosting?

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The control sample was stored at 2-8 deg C and teh stress induced sample did show slight turbidity - that possibly could be due to denaturation. does denatured protein show a different absorption maxima?
could it be that the protein aggregates formed due to stress condition led to masking of sites/residues required for action of reagents in lowry's estimation or for spectrophotometric reading?
si there any method to estimate total protein in such stress induced samples?

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actually, if your sample is turbid it will initially exhibit a higher absorbance due to light scatter. it will, however, continually drop over time. it will stabilize when clear but will be at a lower concentration than the original sample.

do you clarify the turbid sample prior to reading?

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hi swapna,
i cant exactly predict what is the problem in your situation. i wud like to suggest you something, that is
take your protein of interest and boil it for 5 min then check the protein conc by ur traditional way(280 spec and lowry). this could give the idea whether your protein under denatured conditions it will give different readings (less than your control)?
if you get same reading then i wud doubt that your protein is undergoing some process where it is no more detectable (for example lowry's method of detecting protein is based on aromatic aa composition of a protein. may be understress conditions these aa in your protein are getting modified so that u cant estimate the protein, but in this case you 280nm estimation should work fine)
if you have information about above situation, then i wud think that temparature is bringing some irriversible reaction in your protein which is preventing your protien being detected.
if u think this cud be the case
try to induce stress at different temparatures (gradual increase) and estimate the protein conc this exp give you answer if your protein detection is getting affected because of temparature or not?

i hope i did not confuse you
let me know if you need clarification
gud luck
sravan

[
quote name='swapna' date='Mar 13 2006, 07:39 AM' post='44015']
the concentration of a protein sample before and after being subjected to temperature induced stress conditions are different. the concentration of the stress induced sample is lower than the control sample. why is it so?
Concentration was measured spectrophotometrically at 280 nm as well as by lowry's estimation.
[/quote]

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Well, i hd taken a scan of the control and stress samples from 250 - 400 nm without clarifying the stress sample. The OD values on either side of teh abs. maxima is higher for the stress sample in comparison to taht of the control. However, at 280 nm, the OD is less n the stress sample than the control (as predicted by you). so all teh higher values could be attributed to light scattering?

If stress sample OD is measured after clarifying it, value would in any case be lower than the control, i suppose.

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probably and yes.

you might want to try clarifying the control, as well.

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