GAPDH activity assay - (Mar/09/2010 )

Hi,

Does anyone have a protocol for a gapdh activity assay, either for purified gapdh or directly with cell lysates?

I know there is a protocol in Methods of Enzymatic Analysis by Bergmeyer, but I don't have easy access to that book.

Thanks.

jaknight on Mar 9 2010, 05:19 PM said:

Try googling "GAPDH activity assay"

Here's a paper with methods and links to references in the methods used. Looks pretty quick and simple.

http://www.biochemj.org/bj/345/0467/bj3450467.htm

Did that, and I read that paper.

They reference the Bergmeyer book, which I can't get a hold of.

That paper and others give some of the details about the protocol, but I was hoping someone might have something more detailed.

jaknight on Mar 9 2010, 08:17 PM said:

Hi jaknight,
I'm so sorry, I took a closer look at that paper, then tried to get the Bergmeyer protocol but ran into brick walls. I will let you know if I come across something more useful.

Denny on Mar 9 2010, 08:35 PM said:

Check your PM

here is the assay as presented by worthington:

g-3-pdh.

also

i used to assay g-3-pdh many years ago and have the assay in my methods book. let me know if you want me to post the procedure.

Thanks for the responses.

I found a detailed protocol on Sigma's website. It's available here in case anyone in the future is looking for GAPDH assays (http://www.sigmaaldrich.com/catalog/ProductDetail.do?D7=0&N5=SEARCH_CONCAT_PNO|BRAND_KEY&N4=G2267|SIGMA&N25=0&QS=ON&F=SPEC)

The worthington assay looks slightly less complicated. So I'll try that one if this is a no go.

the protocol given by sigma is the reverse reaction.

the assay is more complex and expensive than the forward reaction.

for one thing, the reverse reaction uses nadh in the assay while the forward reaction uses nad. nadh is more expensive and less stable than nad.

for another, the reverse reaction requires linking with another enzyme. you will have to determine how to prevent that reaction from being the rate limiting step (well, it's probably already been done, hence the assay presented).

my vote is for the far less complex and less expensive and more stable forward reaction.

and, yes, i have done both.

I'm encountering the same issue...the protocols posted to measure GAPDH activity are based on cuvettes. And I'm running the assay on a 96 well plate. Has either Jaknight or Denny tried re-formatting on a well plate? I've been having serious issues with getting a decent curve in measuring the formed product from DL-glyceraldehyde.
Thank you, it would be a huge help~!

you should be able to perform the assay in a plate by adjusting the scale. the problem is that the reaction is relatively rapid and you need to read immediately after starting the reaction (we used to follow the reaction by charting the change in absorbance directly from the spectrophotometer).

you could run the reaction and kill at a specific time but one product (nadh) is labile so you'll need to find a way of killing the reaction without causing decomposition.

you can prepare the d isomer from the mixture with dowex-50 (see the link in my previous post)