User:Zachary I. Mendel/Notebook/Zacks Notebook/2013/09/25

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SDS-Page Gel
SDS-Page Gel
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Revision as of 18:32, 1 December 2013

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Objective

To 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.

Description

We 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

  1. Prepare the Gel and Assemble the Electrophoresis Cell
    1. Remove comb and tape from the gels
    2. Rinse the wells with running buffer
    3. Assemble the electrophoresis cell (note diagrams in manual)
    4. Fill the inner and outer buffer chambers with running buffer
  2. Prepare and Load Samples
    1. You prepped your samples yesterday
    2. Heat your samples for 5 minutes at 100C (in the thermocycler)
    3. Load 20uL of protein ladder into column 1 of your gel
    4. Load 20uL of your samples into the appropriate lane of your gel
  3. Perform electrophoresis
    1. Run for 30 minutes at 200V (I need to make sure our power source can do this)
  4. Develop/Stain your gel
    1. Place gel in Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes
    2. Place gel in Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 1 hour
    3. Place gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes
      1. Repeat this step with fresh destain solution 2 more times

Notes

Table 1: Samples in Wells

Well Numbers Sample
1Hemoglobin
2Pepsin
3Pepstatin
40.5hrs Pepsin
50.5 hrs Pepstatin
61 hr Pepsin
71 hr Pepstatin
81.5hrs Pepsin
91.5 hrs Pepstatin
102hrs Pepsin
112hrs Pepstatin
12Hemoglobin

SDS-Page Gel


Absorbance Spectrum of Pepsin with Hemoglobin Image:Hemopepsin09251013zem.png


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

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