MMP zymography

For concentration of proteins in solution, to be followed by SDS-PAGE, I have used acetone precipitation. The protocol is essentially the same as for ethanol precipitation and works very well. However, both methods result in a pellet that can be very difficult to resuspend using aqueous medium, like a Tris-buffered solution. I have done a lot of work with CM that requires concentration and my preferred method is centrifugal concentration with spin filters. Millipore has a good selection of MWCOs and sizes. For large volumes of CM, I use the 70-ml size. Using a 30K MWCO (I am only interested in IgG) 70-ml filter, I can concentrate 70 ml CM down to 1.5 ml in about 30 minutes. Everything stays in solution so no problem with resuspension or denaturation. One caveat is that you must start with serum-free medium when making CM. If you use FBS-containing medium, the concentration of albumin and other serum proteins will be so high that almost any concentration method will result in a gelatinous blob.

Also, we routinely store CM at -80°C (or 4°C if you keep it sterile) prior to concentration with no problems. However, we are not looking at MMPs so I can't speak to their stability.

Hi,

I am planning to use ethanol precipitation to concentrate my samples from conditioned media (CM) based on method by Albini et al. (1995) [Int. .J. Cancer: 61, 121-129 (1995)]. According to this paper, the CM was diluted 1:5 with absolute ethanol and incubated at -20°C for a minimum of 2 hr in order to precipitate the protein. Precipitates were pelleted by centrifugation at 10,000 g for 20 min at 4”C, then the pellet was air-dried and subsequently solubilized in 100-200 ul of 40 mM Tris, pH 7.5.

Has anyone used this method before to concentrate their protein samples ? I wonder whether their ratio of 1: 5 (sample: absolute ethanol) is good or can I actually reduce the ratio to 1:3 or 1:4?

Does anyone of you encounter problem with ethanol precipitation i.e: native MMP activity was affected by ethanol etc?

On top of that, can the CM be first stored at - 80 C and thawed later for precipitation or should I proceed straight to precipitation after collection of CM? After redissolving the pellet in 40 mM Tris pH 7.5, can I store it at -80 C back and run the zymographic experiment the next day?

Thank you very much and I would really appreciate any comments and advice.

Best regards,

Kitson

You're very welcome. Good luck with your project.

Thank u so much, BlueDevil

Original Message
Date:12/06/2012 07:31:00
Thanks alot BlueDevil for your kind input.

But I wish to give counting by cell number a try. Do you have any idea on how it is being performed? Can you please share in great detail? Thanks once again

KItson

The simplest method to count cells is with a hemacytometer. Abcam has a detailed description of how to do this at this Link. Nexcelom sells a disposable hemacytometer that is somewhat easier to use than a glass one.

Thanks alot BlueDevil for your kind input.

But I wish to give counting by cell number a try. Do you have any idea on how it is being performed? Can you please share in great detail? Thanks once again

KItson

Original Message
Date:12/06/2012 02:28:00
Hi everyone,

This forum on zymography is certainly an interesting one.

Does anyone have experience in standardizing the conditioned medium by cell number? I've read a few paper and it seems to me that the amount of conditioned medium mixed with the sample buffer prior to loading to the well is dependent on the cell number.

How does that work? Can anyone share their experience?

On the other hand, is it reliable to determine the amount of protein in the conditioned medium via Bradford/BCA and from there, we normalise the amount of conditioned medium to load to each well?

Thank you very much,

Kitson

We standardize by protein content of the conditioned medium (CM). Standardizing by cell number is a bit trickier since the number of cells will change from the beginning to the end of the time period in which the medium is being conditioned. If you count at the beginning, you won't know how many cells will be present at the end; if counted at the end, the number of cells in the beginning is not known. With total protein content, it doesn't matter how many cells are present. Also, you can standardize by protein content whether the CM is used neat or is concentrated first.

Hi everyone,

This forum on zymography is certainly an interesting one.

Does anyone have experience in standardizing the conditioned medium by cell number? I've read a few paper and it seems to me that the amount of conditioned medium mixed with the sample buffer prior to loading to the well is dependent on the cell number.

How does that work? Can anyone share their experience?

On the other hand, is it reliable to determine the amount of protein in the conditioned medium via Bradford/BCA and from there, we normalise the amount of conditioned medium to load to each well?

Thank you very much,

Kitson

Meant to write "there are many helpful suggestions..."

Basically, what you are saying is that your samples worked before but now they don't. It could be one or more of many different things. A bit more detail would be helpful. Also, have you read through the posts in this thread? There are mant helpful suggestions and links to articles.

Hi everyone,

I stumbled upon this discussion board in hope to troubleshoot my zymography.
So after I stained and destained my gel, there were clear bands against a dark blue background but only for less than a quarter of the gel! From there onwards it is only dark blue with no bands whatsoever for all samples. I am confident that the protein ran to the bottom of the gel because of the indication from the loading dye.

I was thinking that maybe there is something wrong with my samples? But I have used them before and worked beautifully last year. Please advise :(

That's good news. You might be interested in the article at this Link. The title of the article is: MMP Activity in the Hybrid Layer Detected with in situ Zymography. Here's a quote:
Since endogenous dentin MMPs are bound to collagen fibrils in mineralized dentin
(Mazzoni et al., 2009), acid-etching causes MMP exposure, and the subsequent
application of acidic monomers in etch-and-rinse or self-etch adhesives
increases the enzyme activity (Mazzoni et al., 2006; Nishitani et al., 2006;
Tay et al., 2006; De Munck et al., 2009; Osorio et al., 2011).

If you want a pdf of this article, post again.

Thanks for the update.

Hi!
I finally acquired bands of MMP 2 and 9 on gelatin gel. But the thing is; I didn't do anything different than my previous protocol which normally didn't work. All I used was a dental chemical that my dentist friend recommended, she said that some adhesives used in dental procedures enhanced the activity of certain enzymes in the tooth, so I purchased %37 H3PO4 gel (a blue gel commercially available) and directly applied it on my tooth samples, kept them in dark at 4 degrees for 30 minutes, and washed with ice cold acetone for 3 times. washing procedure is like this: add 1ml of acetone for 1gr of dentin powder, mix well, centrifuge at 5000g for 3 minutes, discard supernatant. The rest is protein extraction and gel running.

I just sent it. Post again if you don't get it.

My email is carmemsartorio@gmail.com
Thank you very much in advance
Carmem

I'll probably need your email address.

Dear Dr.
If you could send me this article I would be great.
I am just restarting my zymography and I would like to review it.
Thank you in advance!
Carmem

The following article is an excellent treatment of the subject of zymography.
Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors
Patricia A.M. Snoek-van Beurden and Johannes W. Von den Hoff
BioTechniques 38:73-83 (January 2005)

Let me know if you want me to email the pdf to you. I have other good articles and protocols also.

Dear Cousa

Have you managed to optimize the zymography to get significant bands. if possible can you share your protocol.
Thanks

Hi!
Thank you very much for the paper, I could access it and it looks very useful. I'll try to optimize my protocol according to this one.
My positive control is trypsin, but I'm thinking about purchasing commercial MMP 2 and 9.
Again, thanks a lot, you were very helpful!

I hope it helps. Good luck on your project.