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spectrum of riboflavin

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spectrum of riboflavin

Postby biology_06er » Fri Apr 13, 2007 11:35 am

Hi there

Can someone pls help me with the following questions...we measured riboflavin at diff. wavelengths as well as DNA and nucleiotides.

1-what additional problems are present in obtaining spectrum below 250nm..is it just the fact that below 250 all light is absorbed and hence no light is transmitted meaning no absorbance

2-how do i calculate the DNA conc. and the % of protein present..i have a formula=[protein]/[nucleic acid]x[protein] and then xed by 100. but not really sure what how to firstly calculate the protein and Nucleotide conc. (is that the same as nucleic acid)

3-how can UV spectroscopy be used to get more accurate values for protein concs..as it got something to do with using longer wavelengths??

Thanks heaps,
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Postby biology_06er » Sat Apr 14, 2007 12:53 pm

where is everyone these days!!

can someone please help me??...I seriously have no idea how to work out the above :(
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Postby blcr11 » Sat Apr 14, 2007 4:01 pm

Try here: http://www.biotek.com/products/tech_res ... .php?id=43

So the experiment was to emperically determine at least the absorption spectrum, if not the molar absorptivities, of riboflavin, DNA and protein by taking absorption measurements at various wavelengths, I take it. From those data you should have some feel for the wavelengths at which these molecules absorb most strongly. Those wavelengths can be used to estimate concentrations of material in solution if you know the absorptivity at the particular wavelength. There are "standard" absorptivities assumed for DNA or protein (often called extinction coefficients)--see the above link.

Many molecules absorb strongly in the 150-210nm region--notably, peptide bonds at 190nm. It may be possible to estimate protein concentrations using the absorption at 190nm, but it is far more common to use either a dye binding assay, or the absorption at A280 to estimate protein levels.

Your formula in 2 can't be correct. As written it reduces to 100/[nucleic acid]. You're not far off, but you need to change the denominator. You may think of another way to do it after reading the information in the link above. An additional warning: if you are going to estimate the %P and %N by measuring the concentrations of P and N directly, make sure you use the same units for each measurement--be it ug/ml or mg/ml or mM, or whatever.
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Postby biology_06er » Sat Apr 14, 2007 11:07 pm

H5ey there

Thanks for the reply!...I had a quick read through the link but will have a proper look soon....just another thing I forgot to mention, it says "the nomograph supplied enables you to calculate protein and nucleic acid concentrations from measurements of absorbtion at 260nm and 280nm-does either sample contain protein--calculate the dna conc. of the standard calf thymus dna preparation and te %of protein present..

so is it says calculate shouldnt i be using a formula of something-if i use the nomograph is this how it works...theres a column of protein mg/ml (from 0-2) a wavelegth column of 280 and 260nm with diff. absorbance readings and a N.A mg/ml column (from 0-052)

sooo in order to find amount of protein % present do i just draw a line from the whatever absorbance I got for 280 (As that was the wavelength at which the most absorbance took place) and draw a line and wateva the line corresponds tois that the protein/N.A conc. and this is the amount I will use in my formla?? and as for the % of protein present is it [protein@280]/[N.A@280)*100

but going back to finding the concentration first do I use (@280nm) c=extinction coefficient (the standard one from the site I see it's 1?-is this right?)*OD (which is the absobance I take it)

and thank you sooo much for answering!
much appreciated
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Postby blcr11 » Sun Apr 15, 2007 5:27 pm

I hope I’m not sending you on a moderate wild goose chase referring you to A260/A280, especially if it hasn’t been brought up in your course. The A260/A280 ratio is a measure of the purity of a nucleic acid sample. It isn’t particularly sensitive, but you can read off the approximate %P from the graph.

If you’ve prepared a nucleic acid sample—say a plasmid prep—typically you’d assume most of your sample is NA and assume that at 260 nm a 1 mg/ml solution would give you 20 A in a 1 cm pathlength, corresponding to an extinction coefficient of 20 A/1mg/ml NA. Say you diluted your sample by a factor of 5 to take the measurement and got an A260 of 0.5. By Beer’s Law: A260 = (1/DF) x extinction x concentration. Plugging in the numbers and solving for concentration gives 0.125 mg/ml NA, which is a reasonable concentration for either a high-copy plasmid, or for a midi-/maxi-prep. Even if you have significant protein contamination, (which you should not have if you’ve done the prep correctly) the estimate of NA concentration isn’t far off since the extinction coefficient of protein at 260 nm is 0.5 or about 40 times weaker than NA. The A260/A280 ratio then tells you something about the quality of your prep. It should be close to 1.8 or slightly greater for a pure DNA prep not contaminated with RNA (RNA contamination would give ratios above 2) or protein. If it is 1.7, for example, your prep may contain as much as 55% protein—eyeballing the chart in the link.

Trying to estimate the protein concentration in a NA prep using the A280 is going to be difficult since the NA still absorbs strongly at that wavelength—even more strongly than protein by a factor of 10. I don’t know why you would need to know the exact protein concentration contaminating a nucleic acid prep, but I could see wanting to know how much NA is contaminating a protein prep because you might want to correct the A280 for the cross-over absorption due to NA.

These are general points concerning proteins and nucleic acids. I don’t know if these are the points your professor want’s you to take away.
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Postby biology_06er » Sun Apr 15, 2007 10:21 pm

Hey there...

Umm actually the 260/280 ratio was never brought up in class... but I think I'm kiiinda getting an idea of how to answer the q's. Ill give a quick gist of the experiment carried out...

First, in order to get familiar with spectrophotometer we put 3.5ml of riboflavin in a cuvette and using phosphate buffer as a blank measured the absorbance of RF _at_ diff. wavelengths.

then 2nd par was measuring absorbance of a ALREADY prepared DNA sample at diff. WL's and then again with a sample of pre-prepared nucleotide sample.

and then we had the q's as above...hopefully that gives you an idea of what kind of stuff we had to know...

cheers
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