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protein and carbohydrate assays. analysing absorbance and concentration

protein and carbohydrate assays. analysing absorbance and concentration - ELISA Assay Forum

protein and carbohydrate assays. analysing absorbance and concentration - ELISA Forum. Discuss and post questions including optimization, troubleshooting, protocols and theory of ELISA assays using monoclonal, and polyclonal antibodies or peptides.


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Old 02-07-2009, 12:29 AM
Pipette Filler
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Default protein and carbohydrate assays. analysing absorbance and concentration



Hi everyone!

I'm afraid i need some help again

i did a practical in october last year and teh report is due on monday. i've done the intro, method, apparatus but i am stuck on my results...

We were in teams of 2 and while i did the physical stuff my partner wrote everything down... however, for the last week i have not been able to contact him at all so he can explain the table of number that i have in front of me!

The practical was about alginate and how the alginate lyase gene can be broken down. we had to take the alginate lyase gene Aly from Klebsiella pneumonia in insert it into cosmid vector to produce pSP1. we used e.coli DH5alpha as teh source of pSP1.

This is a brief 8 step objectives:
1. isolate plasmid vector (pGH327) and pSP1 and purify from e.coli.
2. cut pHG327 and pSP1 with restriction endonuclease HindIII.
3. fragments of pSP1 added to cut pHG327 to allow recombination and then treated with dna ligase to reform phosphodigester linkages
4. bacterial colonies screened on MacConkey agar ti detect transformants
5. recombinants with aly gene isolated by detecting expression of alginate lyase enzyme
6. dna isolated from recombinanats, cut with retriction endonucleases adn analyse fragments on agarose gel electrophoresis (allows aly gene to be mapped on pSP1
7. PCR used to identify hindIII fragment of pSP1 with teh aly gene from bacterial extracts
8. expression of alginate lyase by recombinants will be quantified by measurement of specific enzyme activity

i am having 2 problems...

I have 2 gels from electrophoresis. I have measured how far each fragment has moved, but i know i need to do something else with this data, possibly to do with molecular weight?
Does anyone know how i can analyse this data, if i need molecular weight and if i do how i get it?

second problem is from the assays, we did a lyase assay and a protein assay. i have the levels of absorbance for each but i dot knwo what esle i need.... someone said i need to know the concentrtion of the glucose.....

for each assay we had 6 samples of 3 repeats. adn 3 sets of samples. S, C and G1 (not sure what they mean as my "partner" wont tell me...

i can get teh avergae absorbance and teh standard deviation for each sample set... but at teh moment thats about it... i ahve a table of data with an average and SD in it...

I ahve also been told i need to do a trend line on a standard curve,,b ut i'm gettting so very confused!

I dont know if i need to provide any more information, but if i do please ask and i will try my damn hardest to get it if i have it

any help with any of this would be great, i ahve been searching for hours without success...

if anyone wants to see the data or gel photos please ask

is there anyway i can repay someone for their help on here? send beer or flowers lol??

i hope to hear from someone soon. if it makes no sense please tell me and i will re-write

Rach x x
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absorbance , analysing , assays , carbohydrate , concentration , protein


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