User:Daniel-Mario Larco/Notebook/AU Biodesign Lab - 09/03/2013/2013/09/25: Difference between revisions
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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 [http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_1658100.pdf 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 | |||
# Prepare the Gel and Assemble the Electrophoresis Cell | |||
## Remove comb and tape from the gels | |||
## Rinse the wells with running buffer | |||
## Assemble the electrophoresis cell (note diagrams in manual) | |||
## Fill the inner and outer buffer chambers with running buffer | |||
# Prepare and Load Samples | |||
## You prepped your samples yesterday | |||
## Heat your samples for 5 minutes at 100C (in the thermocycler) | |||
## Load 20uL of protein ladder into column 1 of your gel | |||
## Load 20uL of your samples into the appropriate lane of your gel | |||
# Perform electrophoresis | |||
## Run for 30 minutes at 200V (I need to make sure our power source can do this) | |||
# Develop/Stain your gel | |||
## Place gel in Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes | |||
## Place gel in Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 1 hour | |||
## Place gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes | |||
### Repeat this step with fresh destain solution 2 more times | |||
==Data== | |||
From left to right on the image, we have wells 1-12. | |||
-Well 1: Hemoglobin | |||
-Well 2: Pepsin 0.5 hr | |||
-Well 3: Pepsatin 0.5 hr | |||
-Well 4: Pepsin 1 hr | |||
-Well 5: Pepsatin 1hr | |||
-Well 6: Pepsin 1.5 hr | |||
-Well 7: Pepsatin 1.5 hr | |||
-Well 8: Pepsin 2 hr | |||
-Well 9: Pepsatin 2 hr | |||
-Well 10: Pepsin overnight | |||
-Well 11: Pepsatin overnight | |||
-Well 12: Hemoglobin | |||
"Figure 1. Gel electrophoresis" | |||
[[Image:SDS Page Gel JVD 2013 09 25.png|500px]] | |||
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Revision as of 21:20, 30 September 2013
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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
DataFrom left to right on the image, we have wells 1-12. -Well 1: Hemoglobin -Well 2: Pepsin 0.5 hr -Well 3: Pepsatin 0.5 hr -Well 4: Pepsin 1 hr -Well 5: Pepsatin 1hr -Well 6: Pepsin 1.5 hr -Well 7: Pepsatin 1.5 hr -Well 8: Pepsin 2 hr -Well 9: Pepsatin 2 hr -Well 10: Pepsin overnight -Well 11: Pepsatin overnight -Well 12: Hemoglobin |