User:Zachary I. Mendel/Notebook/Zacks Notebook/2013/09/25
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ObjectiveTo run SDS-PAGE to observe amount of hemoglobin remaining after yesterday's digestion. This should be a complimentary measurement to our UV-Vis analysis from yesterday. DescriptionWe will be using a Bio-Rad Mini Protean system with pre-cast Mini Protean TGX gels. The manual for this system can be found here We will be running SDS-PAGE followed by gel development with Coomassie Blue staining. Below is a short description of how we will proceed. Please refer to the manual for more detailed instructions
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Notes
Table 1: Samples in Wells
Well Numbers | Sample |
1 | Hemoglobin |
2 | Pepsin |
3 | Pepstatin |
4 | 0.5hrs Pepsin |
5 | 0.5 hrs Pepstatin |
6 | 1 hr Pepsin |
7 | 1 hr Pepstatin |
8 | 1.5hrs Pepsin |
9 | 1.5 hrs Pepstatin |
10 | 2hrs Pepsin |
11 | 2hrs Pepstatin |
12 | Hemoglobin |
Absorbance Spectrum of Pepsin with Hemoglobin
Corrected Absorbance Spectra of 20μM Pepstatin with Hemoglobin
[[Image:Pepstathemogl09252013zem.png]
For our gel we placed it in 10 mls fixing solution. We then dumped out the fixing solution and stained it with 10mls of a staining solution. This stayed on for 1 hour