How do I prepare a sample to quantify a certain protein with ELISA? - (Jan/08/2007 )
Hello all, can anyone help me?
I would like to use ELISA to determine the amount of a certain protein in a seed. I have a good antibody for my protein and use it for westerns regulary. How would i prepare a sample for loading onto the ELISA plate? Do i need pure protein? Could i just extract total protein and if so, which extraction buffers would i use which would be ELISA suitable?
Thank you for any help, I am a new PhD student so have a lot to learn! 
I am not familiar with seed protein extraction, but in general you can use your total protein extract, but pure is always better because you might get much higher background with the total extract. I would try the total protein extract first, since purifying takes more time/materials/etc. Make sure you do a titration on the plate to determine the optimal dilution for your extracted proteins. Your antibody that you use for western should be able to be diluted out further for ELISA, but you should titrate that as well, or if it is commercially available ask the customer service department for dilution guidelines. It will usually be between 1/10,000 and 1/100,000.
Thanks for your reply..
another question, i use a certain common extraction/loading buffer to run my proteins on SDS gels. could i use this buffer (minus the blue dye) to extract total protein, and then concentrate it down to get rid of the liquid.
I would then need to suspend the protein in a buffer for the ELISA plate, can you recommend one?
For coating plates I use: 13mM NaCO3, 37mM Na2CO3 at pH 9-9.5. You can keep it at RT, but it is a good idea to filter it if you are keeping it for a long period of time.
I would like to use ELISA to determine the amount of a certain protein in a seed. I have a good antibody for my protein and use it for westerns regulary. How would i prepare a sample for loading onto the ELISA plate? Do i need pure protein? Could i just extract total protein and if so, which extraction buffers would i use which would be ELISA suitable?
Thank you for any help, I am a new PhD student so have a lot to learn!
Have you considered a non-ELISA method, such as a straight protein quantitation? How were you planning on making your standard curve on the ELISA?
As Swanny say without a standard your ELISA will only be semi-quantitative.
WAstate is suggesting you coat your protein onto the ELISA plate (which has a high protein binding capacity) then detect with your specific antibody and a species specific secondary antibody conjugated to the enzyme. If you can biotinylate your antibody/have a second antibody raised in a different species you could perform a sandwich ELISA by coating the first/non-biotinylated antibody to the plate, blocking, adding your sample and the detecting with the second/biotinylated antibody and a species specific antibody/streptavidin conjugated to the enzyme.
I noticed you said your antibody works well in Westerns. It might not be suitable for ELISA though as it might not recognise the native conformation of the epitope. If it works in IPs it will work in ELISAs.
Good luck,
Ceri
WAstate is suggesting you coat your protein onto the ELISA plate (which has a high protein binding capacity) then detect with your specific antibody and a species specific secondary antibody conjugated to the enzyme. If you can biotinylate your antibody/have a second antibody raised in a different species you could perform a sandwich ELISA by coating the first/non-biotinylated antibody to the plate, blocking, adding your sample and the detecting with the second/biotinylated antibody and a species specific antibody/streptavidin conjugated to the enzyme.
I noticed you said your antibody works well in Westerns. It might not be suitable for ELISA though as it might not recognise the native conformation of the epitope. If it works in IPs it will work in ELISAs.
Good luck,
Ceri
Thank you for replies, I can purify the protein in question so I will be able to create the standard curve okay, and also my antibody only works on the native conformation; i can not use reducing agents in my buffer for SDS page etc. The problem is extracting all of this protein from each sample, its proving tricky without losing some in the process, which is why i hought it might be easier to extract total protein from a wheat grain and load that onto the ELISA. So i would suspend the dried protein in the carbonate coating buffer?
i'm not really sure about the sandwich ELISA or not stuff, can you tell me the advantages or disadvantages associated with each?
Thanks so much for this help.
I would like to use ELISA to determine the amount of a certain protein in a seed. I have a good antibody for my protein and use it for westerns regulary. How would i prepare a sample for loading onto the ELISA plate? Do i need pure protein? Could i just extract total protein and if so, which extraction buffers would i use which would be ELISA suitable?
Thank you for any help, I am a new PhD student so have a lot to learn!
Have you considered a non-ELISA method, such as a straight protein quantitation? How were you planning on making your standard curve on the ELISA?
Could you give an example or a good reference point for 'straight protein quantitaion'? as i have said in reply to ceri, its really tricky isolating all of my protein in the sample.
It is a possibility that once you dry your protein that it doesn't fold back into the native state. Have you tried drying it before you did your westerns? If that worked, then it will work fine for an ELISA. Sandwich ELISAs are when you coat the plate with an antibody then add your protein on top of the antibody so they bind. Then you come back on top of your protein with another antibody of a different species host, such as a monoclonal coat antibody and a polyclonal top antibody. They have to be different species unless you have a biotinylated version to use as your top antibody. Then you add an HRP conjugate and detect. I use OPD, but a lot of people use Alkaline Phosphatase and TMB. I don't think it matters. I think it's just whatever people are used to. If you need a more detailed protocol and buffers let me know.
As WAstate says the sandwich ELISA uses an protein specific antibody coated onto the plate to capture the protein in the sample and a different species (or biotinylated) protein specific antibody plus a enzyme conjugated streptavidin or species specific antibody to detect it. In a direct ELISA the protein is coated onto the plate and the detected as above.
If you have a complex mixture of proteins the direct ELISA may be less specific. A sandwich ELISA should be more specific in this case.
You talked about using a total cell extract in which case you may be better off using a sandwich ELISA. So you need to have a second protein specific antibody from a different species or to biotinylate some of your first antibody (you can get commercial kits to do this). If this is a lot of hassle start with the direct ELISA coating the protein extract onto the plate and see how you get on. It might be worth thinking about negative controls to see how much background you are getting (e.g. a negative plant tissue, knockout seeds?, seed extracts deleted of your proteins using your antibody and protein G sepharose or seed extracts from a different but related species of plant).
All the best,
Ceri