Assays


So. Friday. I lived in lab. I performed two varying substrate assays and two varying DMSO substrate assay. It was pretty straight forward. After I did varying substrate assay test, I took the values from the two different varying substrate assays and found their change. After the change was found from the two trails of assays I found the average of the change and found the Standard Deviation for the values. The enzyme amount used in the varying substrate was 60 ul of the 20 ng/ul Enzyme. When making solutions of my CA7 Protein in Tris Buffer, I would allow my protein to un-thaw, but as soon as I put it in the centrifuge tube, it would go directly back into the -4 degree refrigerator. The different concentrations of Substrate were 1.5,1.5,2,3,4,5,6,7 mM. The graph began to stabilize with the Standard Deviation off at about 3 mM and this was the concentration used in  the DMSO assay. When looking at the graphs, you want to look at the place where thing begin to stabilize or decrease when adding the variant amount of substance whether its enzyme, substrate or DMSO. The DMSO assay only had varying amounts of DMSO (enzyme, PNPA is constant) as well and I was looking for that same stabilizing image to give me a proper DMSO value to use in inhibition assay. After adding 3.2% DMSO to the solution it begins to affect the function of the protein and substrate and the change in ABS values become small, and this is not what is desired. A functional protein is a must. Note: still done at the 15 sec 5 minute readings at the wavelength 348 nm. (Click the IMAGE to get a full view) Note: the Average ABS value is the Average for the Change of ABS ValuesAlso, when doing assays, I think a little trick is to add the Tris first, water, protein, and then the PNPA (DMSO last if doing the DMSO assays) When adding the PNPA before the protein, it could not allow the substrate to function in the active site as well.  That may not be 100% true, but at least add the solution together a little before OD to let everything work together. TIP: When doing Standard Deviation or my acronym, (Finding the STD (Standard Deviation) LOL! Not Funny :]  ) Anyways, On Excel 2007, go to the Layout Tab, go to error bars, more error bar options, then to custom on the bottom of page, and choose the calculated standard deviation values as the positive and negative values. Delete the horizontal error bars,  by clicking on the horizontal bars on the graph and pressing delete.

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One Response

  1. I wish my graphs looked like that :(!

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