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查看更多产品信息 Novex™ Zymogram Plus (Gelatin) Protein Gels, 10%, 1.0 mm - FAQs (ZY00100BOX, ZY00102BOX, ZY00105BOX)
30 个常见问题解答
在上样前对样品进行短暂加热，能使一些小蛋白酶的结果更好。这是因为加热可使蛋白酶更好地变性，从而不会消化凝胶上的酪蛋白。这种方法也有助于改善条带的清晰度。
可使用Colloidal Blue染料或SimplyBlue SafeStain对Zymogram凝胶进行染色。无需固定步骤。我们推荐在复性和孵育凝胶以获得酶活性后，使用SimplyBlue SafeStain染色。如果在检测活性较弱的酶时需要更高的对比度，可使用0.5% Coomassie R250染料。
Zymogram凝胶本质上是含有底物的Tris-甘氨酸凝胶。蛋白质标准品的电泳只取决于丙烯酰胺的比例，因此在两种凝胶上的电泳应该是相同的。但是，如果标准品是预染色的，则蛋白质会由于Zymogram凝胶的染色（或预染色）而呈现为不同的颜色。
不应该，样品不应加热或还原，以便复合蛋白酶能以单个单位的形式迁移，便于电泳后复性。样品应包含一半样品和一半2X Tris-甘氨酸SDS样品缓冲液。先将样品在室温下放置10分钟，然后再上样。
基质金属蛋白酶（MMPs）的底物包括：
MMP-1：组织胶原酶：胶原蛋白1、2、3、4、6和10
MMP-2：明胶酶：明胶、胶原蛋白4、5和7
MMP-3：基质分解素2：酪蛋白、纤连蛋白、层粘连蛋白和弹性蛋白
MMP-7：基质溶解因子：酪蛋白
MMP-8：中性粒细胞胶原酶：胶原蛋白
MMP-9：4型胶原酶：明胶
MMP-10：基质分解素2：酪蛋白
MMP-12：金属弹性蛋白酶：弹性蛋白
Invitrogen Zymogram凝胶是对以酪蛋白或明胶为底物的蛋白酶进行检测和表征的良好工具。在SDS条件下，蛋白酶可在含有上述任意一种底物的凝胶上进行电泳。经过简单的复性和染色程序，蛋白酶在黑色背景下呈现为透明的条带。这些凝胶通常可用于检测基质金属蛋白酶——一个与癌症和一些炎性疾病发病机制相关的酶蛋白家族。
Zymogram缓冲液应保存在4℃。
Zymogram凝胶在4℃下可有效保存8周。
我们推荐将Zymogram凝胶保存在4℃。
Zymogram凝胶的浓缩胶部分不含底物（明胶或酪蛋白），但在凝胶灌制过程中可能有少量混入。
包含，Zymogram凝胶包含长度约为8-9 mm的4%浓缩胶。
不同上样孔规格的凝胶的推荐上样体积和蛋白质上样量见Invitrogen预制胶电泳指南（https://tools.thermofisher.com/content/sfs/manuals/electrophoresisguide_man.pdf）第8页。
Zymogram凝胶中丙烯酰胺：双丙烯酰胺的比值为37.5:1，交联剂的比例为2.6%。
我们的Zymogram凝胶不含SDS。我们推荐选择Invitrogen Tris-甘氨酸SDS样品缓冲液和Invitrogen Tris-甘氨酸SDS电泳缓冲液与这些凝胶一起使用。推荐酶活检测使用Invitrogen Zymogram复性缓冲液和Invitrogen Zymogram孵育缓冲液。
Invitrogen 10% Zymogram明胶凝胶是以0.1%源自猪皮的A型明胶（Sigma货号G2500）为底物的10% Tris-甘氨酸凝胶。
Some small proteases work better if the sample is heated for a short time before loading so the protease is denatured better and it doesn't digest the casein on its way through the gel. This may also help to improve the band sharpness.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Zymogram gels can be stained using Colloidal Blue stain or SimplyBlue SafeStain. The fixing step is not required. We recommend staining with SimplyBlue SafeStain after renaturing and developing the gel for enzyme activity. If greater contrast is needed for detecting weakly active enzymes, a 0.5% Coomassie R250 stain may be used.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Zymogram gels are essentially Tris-Glycine gels containing the substrate. Protein standards run based solely on the percentage of acrylamide and hence should run the same in both kinds of gels. It is quite possible though that if the standard is pre-stained, the proteins will appear a different color because of the staining (or pre-staining) of the Zymogram gels.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
No, samples should not be heated or reduced so that multiunit proteases migrate as a single unit that can be renatured after electrophoresis. The sample should be one part sample and one part 2X Tris-Glycine SDS Sample buffer. Let the sample stand at room temperature for 10 minutes and then load the sample onto the gel.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The substrates for the matrix metalloproteases (MMPs) are:
*MMP-1: tissue collagenase: collagen 1, 2, 3, 4, 6, & 10
*MMP-2: gelatinase: gelatins, collagens 4, 5, & 7
*MMP-3: stromleysin-2: casein, fibronectin, laminin, elastin
*MMP-7: matrilysin: casein
*MMP-8: neutrophil collagenase: collagen
*MMP-9: type 4 collagenase: gelatin
*MMP-10: stromelysin-2: casein
*MMP-12: metalloelastase: elastin
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Invitrogen Zymogram gels are an excellent tool for detecting and characterizing proteases that can utilize casein or gelatin as a substrate. Proteases are run on a gel containing one of these substrates under SDS conditions. They can be visualized as clear bands against a dark background following a simple renaturing and staining protocol. These gels are commonly used to detect matrix metalloproteases, a family of enzymes linked to the pathogenesis of cancer and some inflammatory diseases.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The Zymogram buffers should be stored at 4 degrees C.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The shelf life of Zymogram gels is 8 weeks when stored at 4 degrees C.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
We recommend storing the Zymogram gels at 4 degrees C.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Our Zymogram gels do not contain the substrate (gelatin or casein) in the stacking gel although a small amount might mix in during casting of the gel.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Yes, our Zymogram gels do contain a 4% stacking gel that is ~8 to 9mm long.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The recommended sample loading volumes and protein loading amounts for the different well formats can be found at:
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gels/recommended-well-loading-volumes-sample-loads.html.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The ratio of acrylamide:bisacrylamide in our Zymogram gels is 37.5:1 and percentage of cross-linker is 2.6%.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Our Zymogram gels do not contain SDS. We recommend using the Invitrogen Tris-Glycine SDS Sample buffer and the Invitrogen Tris-Glycine SDS Running buffer with these gels. Invitrogen Zymogram Renaturing buffer and Invitrogen Zymogram Developing buffer are recommended for enzyme assays.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The Invitrogen 10% Zymogram Gelatin gels are 10% Tris-Glycine gels with 0.1% Type A gelatin from porcine skin (Sigma Cat. No. G2500), as the substrate.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.