10 replies to this topic

[metaltemujin]

I am working on Beta glucosidase and Galactosidase enzyme assays for checking the anti-diabetic inhibitory potential of my samples. I got the enzymes(Quant:1 Ku) in around December last year. It worked then.

After the first experiment(s) I had to leave for my winter break, so stored the enzymes in 4 degs (all in centigrade/ deg C) (As mentioned in the bottle). But my project guide told me to put it in -20 degrees as I had suspended the entire quantity of the enzyme in 0.1 Molar Phosphate buffe (K₂HPO₄ and KH₂PO₄).

It's been there all the time(around 3.5 months now)

When I use the enzyme, I have a working solution out of it by taking a small volume of it and storing at 4 degrees to use for the next week,etc.

But after the first attempt, the reaction has not worked. Except when I used around 10 KU by mistake with the substrate - This sample was stored at 4 degrees, but when I tried to make the required quantity sample (0.04 units ml^-1), it again did not work.

Only once did I test with my sample, after that I have only been trying the 100% reaction( buffer + enzyme + substrate) of the enzyme Substrate reaction and it is not been working. Working = Buffer + Ez+ Sub @ 37 deg C for 30 mins = Yellow color formation from colorless solution.

The Beta glucosidase, I got in Feb this year and from the first attempt itself its not been working. It's been stored at -20 as well, like the previous enzyme.

Substrate: Beta - D - Gluco/galactopyranoside. (stored at 4 degree Centigrate)
Buffer: Phosphate Buffer 0.1 M (as mentioned above). Fresh batches prepared regularly.

Please help, my project has been stuck for almost 3 months because of this and I am not able to troubleshoot where the assay is going wrong.

Edited by metaltemujin, 15 April 2011 - 05:34 AM.

[mdfenko]

you may have denatured your enzymes by freezing them. you may have to purchase new ones.

read the data sheet to determine the proper way to store them before and after dilution.

[metaltemujin]

The Datasheet says after reconstitution, it is to be stored at -20 degrees.
This is what it said:
"Prior to reconstitution store at +4oC.
After reconstitution store at -20oC.
Storage in frost-free freezers is not recommended. This product should be stored undiluted. Avoid
repeated freezing and thawing as this may denature the protein. Should this product contain a precipitate
we recommend microcentrifugation before use."

Can contamination by pippet tips denature the enzyme? My seniors suggested me this possibility.

[mdfenko]

metaltemujin, on 19 April 2011 - 09:54 PM, said:

Can contamination by pippet tips denature the enzyme? My seniors suggested me this possibility.

that depends on with what your tips are contaminated. not likely if your tips are new and fresh.

[metaltemujin]

Then I am quite lost .... they were from the packet.

[DRT]

Could you be storing the enzyme too dilute? Less than a few ug/mL could give you problems with adhesion to surfaces.

Just to clarify; the yellow reaction implied that you were using a pNP-G based substrate. Is this correct?

As you are looking at anti-diabetic inhibitors would it be an idea to move onto alpha-glucosidase inhibition while you are trying to sort out the problem with the beta-glucosidases? That way at least you will have some results coming in.

[metaltemujin]

I am storing it at 0.01 Molar phosphate buffer, as per the instructions in protocol/Data sheet.

I am considering changing of my protocol to see if it gives any results.

As for alpha-gluc, well, The enzyme-substrates for Alpha is a bit too costly right now, So i am only stuck with beta as of yet. I can up until then do other assays, maybe? Non anti-diabetic ones, like TPC or FRAP, but that wouldn't solve the purpose.

And yes I am using pNP-G based substrates presently.

P.S: I am sorry for the late replies, I thought people dont respond frequently here, hence I check only every few days. will check often from now on.

[mdfenko]

you can be notified when a reply is made by checking the box when posting.

[metaltemujin]

Ah. Still no solution Hmm... i'll have to figure it out myself T_T

[sup1109]

hello there,
i'm doing the same project as yours.how was it?i've just started and would like to ask a few questions. hope to hear from u soon.thanx.

[Eternalist]

Like other posts, my rule is to thaw a frozen enzyme once....maybe twice. I have had good luck by initially aliquoting into subsamples that I will use later then, freezing. When you thaw a vial, dilute and add BSA carrier protein (1%) to prevent the protein from binding to vials... then store at 4 deg C while using that particular vial.