ELISA for cGMP

[strawber]

Hi guys,

I am going to measure the cGMP from tissue and my question is should I dilute my sample first before the measurement? in the manual from the ELISA kit there is no information about dilution for tissue sample. Can anyone help me?

thank you so muuuuuccchhhhh!!

[Elisadeveloper]

What is the tissue, what are the expected concentrations, what is the kit you intend to use. Are you extracting?

[strawber]

The tissue is from the rat's brain, the kit that i will use is parameter cGMP assay. In the manual from the kit it said that the sample require dilution or lysis, and the kit also provide calibrator diluent (for cell culture supernate, serum, plasma, saliva, urine samples) and cell lysis buffer (for cell lysate samples) but there is no information which solution should i used for tissue sample. The expected concentration is 0.190-1.961.
Yes i did extraction from my sample with comparison between the tissue and extraction buffer 1:1.
What should i dooo?? thank you sooooo muuuuuccccch!!!

[Elisadeveloper]

If you are using the R&D systems kit, it should work flawlessly for appropriate samples.

I am assuming that you have an appropriate means of harvesting the brain tissue without loss or degradation of the cyclic GMP. For cells, the kit recommends freeze thaw several times followed by lysis in the buffer they provide. This may be a valid approach for a brain homogenate, providing that you don’t have enzymatic cyclic GMP loss. You may want to consider a quick stability assessment by spiking in cyclic GMP in your homogenate/lysis buffer to make sure it doesn’t degrade rapidly. You could use the kit standard to spike into the homogenate/lysis buffer and make sure that you get the same result with various storage periods and temperatures of the homogenate/lysis buffer. You can also buy cyclic GMP cheaply if you want a alternative higher concentration source (Sigma G6129) The kit insert recommends using the lysis buffer as a calibrator diluent, (rather than RD5-5) if you use the lysis buffer to extract the cyclic GMP. A literature search may allow you to find alternative methods for brain. The following uses homogenization followed by TCA precipitation with the cyclic GMP ending up in the TCA supernatant which is dried down by ether
[Only registered users see links. ]. Simply reconstitute the supernatant extract in the RD5-5 and run the curve in RD5-5 in this case.

The TCA pellet provided a convenient means of determining protein content.

In terms of the appropriate dilution to use, unless you can find a literature report detailing expected concentrations, try neat 3, 10, 30, 100 etc, or some other scheme, and it will be important to demonstrate that your homogenate + lysis buffer dilutes in parallel manner to the curve (in lysis buffer alone) Avoid dilutions that are not parallel when you are analyzing samples or fix the problem. Also spike recovery (spiking cGMP into the homogenate) experiments are useful.

Finally, the technical support provided by R&D systems is excellent, so don’t hesitate to get on the phone and ask them for advice as they probably have data on exactly on what you intend to do.

Good luck!