Rt pcr protocol pdf
If you are interested in knowing the recipes that others have used to rt pcr protocol pdf the buffers needed for gelatin zymography, see our Gelatin Zymography Reagent Recipes page. This protocol has been adapted from a previously published JOVE paper .
If you are not sure how to identify which MMPs are in each band of your developed zymogram, click here to see our post on this subject. Click here to see our protocol on Preparing MMP-2 Standards. Assemble the electrophoresis chamber with the zymogram according to manufacturer’s instructions. Load 20 uL of samples and protein standards into the zymogram wells using gel loading pipette tips. Check for bubbles to ensure that the current is flowing. Keep an eye on the blue dye in each sample to ensure that the proteins are migrating as expected.
The blue dye should reach the bottom of the gel at the end of the run. Carefully score the edges of the gel with the gel knife, and cut away the bottom part of the gel to free it from the cassette. Mark the bottom-right side of the zymogram by cutting away at the corner. The cut should correspond to the leftmost side of the gel while loading samples. Transfer the zymogram to the top of a sheet-protector. Scan the gel to record the position of the colored molecular weight protein standards.
Transfer the zymogram to a container and fill it with 100 mL of Renaturing Buffer. Incubate under gentle agitation for 30 min. Remove the Renaturing Buffer and add 100 mL of Developing Buffer. Remove the Developing Buffer and add another 100 mL of Developing Buffer. Remove the Developing Buffer and replace it with 100 mL of deionized water.
Wash under gentle agitation for 5 minutes. Wash a second time under gentle agitation for 5 min with a fresh 100 mL of deionized water. Wash a third time under gentle agitation for 5 min with a fresh 100 mL of deionized water. Note: Some protocols call for using only 20 mL of staining solution for 1 hour, followed by a couple of washes in water to de-stain the gels.
However, I have found that using a lower volume and shorter staining time period results in a lightly-stained zymogram that is harder to interpret. Transfer the zymogram to the top of a sheet-protector and scan the gel to record the size and position of the clear bands. The bands of clarity indicate regions where MMPs have cleaved the gelatin in the zymogram. The position of the clear bands can be used with the molecular weight protein standard to identify the molecular weight of the MMPs that cleaved the gelatin at that location. See this post for our list of possible MMPs that can correspond to each molecular weight. The size of the clear bands can be measured in ImageJ in order to semi-quantify the relative amount of MMPs in each sample. Detection of Functional Matrix Metalloproteinases by Zymography.