Interested

Several companies offer a beta-lactamase assay kit. This is the technical bulletin for the one from Sigma-Aldrich:

https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/129/727/mak221-tech-bulletin-ms.pdf

It doesn't say what the buffer is, but usually the buffer used for beta-lactamase assays is a sodium phosphate buffer. You can measure the pH yourself with a pH meter. It will probably be in the range 7-7.5.

Phosphate buffer is not suitable for metallo-beta-lactamases (MBLs), however, because it is not compatible with zinc ion that is required for activity of MBLs. I would substitute HEPES buffer when using an MBL enzyme.

Since you supply the beta-lactamase, the enzyme concentration and reaction time are parameters you will have to determine yourself. If you have an absorbance plate reader, this is easy to do. Just make a set of dilutions of the enzyme in the pM to nM range and measure the absorbance as it increases over a period of time for each concentration. Select an enzyme concentratoin and reaction time during which the absorbance increases in a linear fashion (very important) and produces an absorbance signal that is large enough for practical use (at least a change of 0.1 versus the no-enzyme control.)

Hello Adam,

Thank you for your lovely answer.

There is two buffers that used in beta-lactamase assay, hydrolysis buffer which is used to hydrolyse the nitrocefin and create the standard curve, and the assay buffer which is used to assay beta-lactamase activity. Unfortunately, sigma-aldrich and ABCAM doesn`t mentioned what is the buffers. My question, is hydrolysis and assay buffer are same? what pH of each one ?

Beta-Lactamase that I work on now is AmpC (not class B) , can you please help me and confirm what buffers I need to use to assay the activity?

Also, it is mentioned in the technical bulletin that different concentrations of hydrolysed nitrocefin applied in the well-plate and run in kinetic mode with the samples to establish a standard curve , If I want to draw the standard curve, at which minute do I choose the absorbances and plot them versus the concentrations of nitrocefin??

Ammar Ibrahim I imagine this is now not relevant to you since you posted the question some months ago. I have been using Abcam's kit and I have just run out of the assay buffer so was just seeing if it's possible to buy a replacement. The kit was very expensive and I barely got any experiments out of it! For the standard curve, it says that you can just take the end point absorbances because these values shouldn't change over time unlike the samples and positive control. So, if you have the hydrolysed nitrocefin standards on the same plate as your samples, and you run the assay for an hour, then just plot the final time point values for your standard curve. However, I think if you are doing the standards in a separate plate, then you don't even need to run it in kinetic mode - just record absorbance at 490nm. My standard curve is pretty rubbish and I can't even repeat it because I've ran out of the buffer :(

Kits can be very expensive. If you need to do a lot of experiments, it is cheaper (and easy) to prepare the reagents yourself. You only need the enzyme, nitrocefin (https://www.sigmaaldrich.com/US/en/product/mm/484400),

a buffer, and microplates if using a plate reader.

Prepare a concentrated nitrocefin stock solution by dissolving the solid in DMSO. Store the stock solution in small portions in the freezer (-80 if you have one). It will last a long time if stored properly.

For Class A and C enzymes, you can use almost anything for the buffer, such as 0.1 M sodium phosphate buffer at pH 7.0. For some class D (OXA) enzymes, you have to include sodium bicarbonate, at 10-50 mM. For class B metalloenzymes (e.g. NDM) , you should avoid phosphate buffer because you have to add Zn2+. I've used HEPES at pH 7.0 with 1 µM ZnSO4.

If you are using microplates for the assays, you should also include a little bit of a nonionic detergent in the buffer to keep the enzyme from sticking to the plate. I've used 0.005% Triton X-100.

You can buy 96-well clear polystyrene microplates with flat-bottom wells in packages of 25-100 plates. The least expensive ones are not sterile, and not tissue-culture-treated. These work very well. There is a significant cost outlay, but it will save in the end if you need to do many experiments. An example is VWR catalog number 76446-956, 100 plates for about $200.

Adam B Shapiro Thanks for your very informative answer. I did actually use a non-kit nitrocefin previously but this was for disc diffusion experiments. I only decided to use the kit mainly because of the +ve control that it comes with (since I'm not sure how to obtain a beta-lactamase for use as a +ve control - I haven't got enough time left in my PhD to purify a recombinant one). I am actually working with an MBL and did not add zinc in my assays. I will now repeat using your suggestion of HEPES pH 7 + 1uM ZnSO4.

For nitrocefin standard curve, I am confusing what buffer I must use. In the last assay, I used 0.1M of NaOH. The highest absorbance that I got at the end of the kinetic mode was 0.12 which is lower than the absorbance ot the enzyme at the same minute.

Ammar Ibrahim if you are using the Abcam kit: for the standard curve, you use the ASSAY buffer for preparing the standards (NOT the hydrolysis buffer). Only use the hydrolysis buffer for hydrolysing nitrocefin at 60 degrees for 30 min BEFORE diluting with the assay buffer.

Emma Thompson I didn’t use Abcam kit because it is expensive, I am using NaOH as hydrolysing buffer for nitrocefin’s standard curve, and assay buffer for kinetic readings. My problem is with the hydrolysis buffer because I am not sure if I am using the right buffer for the standard curve.