October 15, 2019
Materials and Methods
The first step consisted of occurring 0.5 mL of the sample which was then placed into a 1.5 mL
tube. The cells were then centrifuged for 2 minutes at 13.5 rmp. 20l of supernatant was placed into a
new microfuge tube. Next, 20l of 2X Tris-Glycine Sample Buffer was added to the tube and vortexed.
The tube was then incubated at room temperature for 10 minuets. Following that step 25l of the sample
was loaded into precast gels which were purchased from Invitrogen. The gel was then running with 1x
Tris-Glycine SDS Running Buffer using the standard running conditions which consisted of 125V of
constant voltage. After the gel was finished running Zymogram Renaturing Buffer was diluted 1:9 with
deionized water and the gel was incubated in the buffer with gentle agitation for 30 minutes at room
temperature. When the Buffer was being diluted with the deionized water 100 ml of the deionized water
was used for one or two mini-gels. The Zymogram Renaturing Buffer was then decanted and replaced
with 1x Zymogram Developing Buffer. Next, the gel was equilibrated for 30 minutes at room temperature
with gentle agitation and then replaced with a new 1X Zymogram Developing Buffer. The sample was
then left to incubate over night at 37 degrees Celsius for maximum sensitivity. Next the sample was
stained with Coomassie Blue R—250 for at least 1 hour.