Gelatin zymography/casein zymo electrophoresis kit
Frequently Asked Questions
General Questions
- 【01】 What is Zymography?
- 【02】Why can pro-MMP with no enzymatic activity be detected as a band in zymography?
- 【03】 How long does electrophoresis take at a constant current of 15mA?
- 【04】 What should I do if I want to take a good photo of the gel?
- 【05】 How much protein should I apply per lane?
- 【06】 How to extract MMPs from tissues?
- 【07】 What should not be done in sample processing?
- Q: Do you have information about the effects of anticoagulants used to draw blood?
- Q: What is the size of the precast gel included in the Gelatin Casein Zymo Electrophoresis Kit?
Gelatin Zymography
- 【01】 What is the gel concentration of the precast gel?
- 【02】 What is the band detected between pro-MMP2 and MMP2?
- 【03】Q: Is the mobility of mouse and human pro-MMP9 different?
- 【04】 A band is also detected on the polymer side of proMMP9.
Casein Zymography
General Questions
【01】 What is Zymography?
Zymography is a method of detecting the enzymatic activity of enzymes separated by electrophoresis . When a gel containing substrate proteins such as gelatin or casein is used, the entire gel is stained blue by protein staining, but areas with enzymatic activity are detected as clear bands due to degradation of the substrate protein.
【02】 Why can pro-MMP with no enzymatic activity be detected as a band in zymography?
pro-MMPs are inactive in vivo because the active site is masked by the propeptide sequence. In zymography, SDS treatment releases the pro-peptide sequence that masks the active site, and pro-MMP also exhibits enzymatic activity.
【03】How long does electrophoresis take at a constant current of 15mA?
It depends on the equipment you use, but as a guide, it will take about 3 hours.
【04】What should I do if I want to take a good photo of the gel?
In order to photograph the bands clearly, it is ideal to photograph the gel with white light from below.
When using a gel imaging device, it is almost always combined with a UV transilluminator, but even in that case, a special plate "UV-white conversion screen" that converts UV into white light can be taken by placing it on the UV illuminator.
There are also transilluminators available with white light as well as UV, or with only white light. Along with these, images can be captured using a common digital camera.
【05】How much protein should I apply per lane?
Since the MMP activity contained in the sample varies depending on the type of tissue/cell, it is difficult to generalize the optimum amount of protein to be applied. As one method, apply about 10 μg of protein as a preliminary study, and determine the optimum concentration based on the results.
In addition, when culture supernatant is used as a sample, it is often the case that the liquid volume is used as a standard.
【06】How to extract MMPs from tissues?
Since MMPs are strongly bound to matrix components in tissues, it is generally said that their extraction efficiency from tissues is poor. For the extraction method, the tissue is homogenized with a polytron in a buffer containing 10 mM CaCl 2 and 0.25% Triton X-100, and the supernatant is recovered by centrifugation. Use the supernatant as a sample. Other extraction methods from tissues include solubilization with urea and heat treatment at 60°C. For details, see Woessner Jr.'s paper (Methods in Enzymology Vol.248, pp510-529).
【07】What should not be done in sample processing?
If a reducing agent such as 2-mercaptoethanol or dithiothreitol (DTT) is added to the sample, or if the sample is heat treated, the protein will be denatured and the enzymatic activity will be inactivated.
Q: Do you have information about the effects of anticoagulants used to draw blood?
A paper by Gerlach et al. (Analytical Biochemistry Vol.344, pp147-149) reports a comparative study of serum and plasma using three types of anticoagulants (citrate, heparin, and EDTA). Please see the paper for details.
Q: What is the size of the precast gel included in the Gelatin Casein Zymo Electrophoresis Kit?
Scale illustration of a precast gel plate
【01】What is the gel concentration of the precast gel?
Contains 10% acrylamide and 0.1% gelatin.
【02】What is the band detected between pro-MMP2 and MMP2?
It is intermediate MMP2. MMP2 is activated by cleavage of the pro-peptide portion from pro-MMP2, and Intermediate MMP2 is an intermediate that appears in the process.
【03】Q: Is the mobility of mouse and human pro-MMP9 different?
MMP9 has N-type and O-type sugar chains, and the size of the sugar chain that binds differs depending on the animal species. Therefore, compared to human-derived, mouse-derived is detected on the high-molecular side.
【04】A band is also detected on the polymer side of proMMP9.
Complexes of MMP9 with dimers of MMP9 or other proteins (TIMP, lipocalin, α2-macroglobulin, etc.) are possible.
【01】What is the gel concentration of the precast gel?
Gradient gel with 8-13% acrylamide concentration and 0.05% casein.