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Post by fhorn2 » Sat Jan 29, 2005 3:19 pm

can anyone help me with the following qns?i can onli find the ans to part a but the rest i dunno wads happening...thx a zillion to whoever can help to solve it.. =)

1.Determination of the concentration of an antigen of unknown concentration.

You are using ELISA for this exercise. Antigen-Q is a protein of a single polypeptide chain with a molecular weight of ~235 kDa.

Microtiter wells were coated with a pre-determined amount of antigen-Q, in 100 l of 50 mM sodium bicarbonate, pH 9.0 overnight at 4ºC.

The next morning the solutions in the microtitre wells were discarded and the wells were washed twice with PBS (phosphate buffered saline).

150 l of PBS containing 1% w/v BSA (bovine serum albumin) were added to each well and the plate was incubated at room temperature for 30 minutes.

(a)What is this step for? Why do you use BSA? Could you have used anything else? If so, what would you use?

It had been established that when the monoclonal antibody, from mouse, anti-Q was used at 50 ng/ml in 100 l in the subsequent incubation, the antigen-Q can be detected by a second antibody conjugated with alkaline phosphatase to give a non-saturating activity to convert the substrate pNPP, which can be read at 405 nm with an OD of 0.8.

(b)Could you have used the substrate BCIP/NBT? If not, why not?

You had two samples of antigen-Q preparations, A and B, of unknown concentrations, and a standard preparation of antigen-Q, S, at 0.5 M. You made a serial 2-fold dilution of each of the three samples (10 dilutions each).

(c)How would you do the serial dilution? Please specify volume and what buffer would you use?

You then incubated each of the diluted samples (30 total) with a solution of the anti-Q antibody at room temperature.

(d)How would you carry this out? Please specify volumes, buffer used, anti-Q preparations.
(e)In addition to these 30 samples, you’ll need a couple more mixtures. What would they be?

During the incubation, you washed your microtitre plate coated with antigen-Q twice with 150 l of PBS containing 0.1% BSA for each well.

(f)Should you leave the wells empty to wait for the incubation to complete? or should you leave the wells in the last wash and empty them just before use? Why?

After 2 hours (previously established), you transferred 100 l of the antigen antibody mixture to the coated plates. You incubated the plates, now with anti-Q and the competitive antigens, further for 2 hours at room temperature.

You washed your plate twice with 150 l of PBS containing 0.1% BSA for each well. You then added 100 l of a standard solution of the second antibody conjugated with alkaline phosphatase.

(g)What should the second antibody be against? and in what animal should the second antibody be made?

After incubation at room temperature for 1 hr, the plate was washed again as before. Finally, 100 l of the substrate pNPP was added.

After development, the plate was read. The reading of the 30 samples are given below.

Dilutions QS QA QB
neat* 0.134 0.138 0.863
1 0.132 0.145 0.785
2 0.149 0.297 0.822
3 0.159 0.484 0.831
4 0.251 0.637 0.790
5 0.407 0.754 0.803
6 0.549 0.789 0.788
7 0.694 0.811 0.812
8 0.804 0.792 0.825
9 0.798 0.801 0.799
* neat means no dilution

(h)You were asked in (e) that you would need extra samples. What readings should they give?
(i)How should you plot your data? (May supply you with a hint later).
(j)What is the concentration of antigen-Q in sample A? and sample B? Show calculations where necessary.
(k)If the sample in A has a concentration of 75 g/ml, what can you say about the sample?
(l)If you analyze sample A by SDS-PAGE, how many bands would you see?

thx once again~!!

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Post by ntu_biosci » Thu Jan 19, 2006 1:34 pm

walao.. do u need to post the whole qn paper here and ask other ppl to gv u answer? do urself la..

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Inland Taipan
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Post by MrMistery » Thu Jan 19, 2006 7:50 pm

We don't anser homework questions. It is our policy
"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter

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King Cobra
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Post by Dr.Stein » Fri Jan 20, 2006 6:28 am

I see it as technical questions of such labwork. If you read your labwork protocol/manual and did your labwork well, I am sure you can answer them easily. They are common questions for ELISA labwork. I can make it for you but I won't. That's your assignment.

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