Protein - Protein quantitation (Oct/24/2005 )

Dear forum buddies

I must say whoever created this forum is a 'star', I'm so equipped with knowlegde within a small space of time.

Please help me I want to run my protein on SDS page gel, may one suggest which easy, quicker and reliable method should I use to quantify my protein?

second question is I bought an antibody which was supposed to arrive frozen but unfortunately bcos it was delayed in the airport until it was no longer frozen upon arrival, how do I check the intergrety and whether it is still in good condition.

Thanks
Lyndza

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hi
for quantification of proteins, bradford assay is quick, quite good reproductible and easy ti set up.

For your antibody, you can ask the company to deliver you a new one, if you haven't already use the thawed one. If you can't get a new one or want to use the mentionned, try to use it with in a gradient positive test. You need to load proteins in gradients of quantity 1 2 5 10 15 20 25 30µg of protein (total proteins or proteins of a specific compartment) and apply the antibiotic. You are supposed to see a gradient of intensity of the band.
Second test : with a positive extract, you need to do in a single gel at least 4 assays. You load on each well same quantity of protein. You transfert it on membrane. Then split the membrane in as many lanes as you want to test concentrations. Usually, dilutions used are ranging from 1:200 to 1:5000. So you'll need to test first antibody dilutions 1:200, 1:500, 1:1000, 1:2000, 1:3000, 1:4000 and 1:5000. Then use a secondary antibody at a unique dilution for all pieces of membrane. Apply ECL on each pieces at the same time and for a same moment. Then wrap all pieces in one saran, put in the same cassette, and expose to the same film.
You'll suppose to see again gradient of intensity.

fred

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Hi,

also for protein quantification BCA works well:
http://www.technochemical.com/instruction/ACF1719.pdf

Seb_

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Fred and Tryptopan thanks for elaborative info

Lyndza

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Fred and Tryptopan thanks for elaborative info

Lyndza

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We use a simple serotypical test. If you have access to a microscope, simply mix your antibody and antigen in appropriate ratios on a microscope slide and incubate at appropriate temperature. Theorectically this reaction should occur instantaneously but allow a few minutes. Insert cover slip and observe under the microscope. Aggregates of antibody and antigen will be created if your antibody is still active. If not you will probably see nothing depending on the size of your antigen. If you are using a tagged antibody this may not be useful but I hope it helps.

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