User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/09/23: Difference between revisions
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Procedure followed as described by [http://openwetware.org/wiki/User:Matt_Hartings/Notebook/AU_Biomaterials_Design_Lab/2013/09/25 Dr. Hartings on Sept. 25 2013] | Procedure followed as described by [http://openwetware.org/wiki/User:Matt_Hartings/Notebook/AU_Biomaterials_Design_Lab/2013/09/25 Dr. Hartings on Sept. 25 2013] | ||
=== Prepare the Gel and Assemble the Electrophoresis Cell=== | ===Electrophoresis=== | ||
=====Prepare the Gel and Assemble the Electrophoresis Cell===== | |||
# Remove comb and tape from the gels | # Remove comb and tape from the gels | ||
# Rinse the wells with running buffer | # Rinse the wells with running buffer | ||
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# Fill the inner and outer buffer chambers with running buffer | # Fill the inner and outer buffer chambers with running buffer | ||
Note: 200mL of running buffer (10x SDS-PAGE running buffer 30.3 g Tris, 144.1g Clycine, 10g SDS, water to 1L) were diluted to 2 L. This amount was necessary in order to fill up the inner and outer buffer chambers | Note: 200mL of running buffer (10x SDS-PAGE running buffer 30.3 g Tris, 144.1g Clycine, 10g SDS, water to 1L) were diluted to 2 L. This amount was necessary in order to fill up the inner and outer buffer chambers | ||
===Prepare and Load Samples=== | =====Prepare and Load Samples===== | ||
# Five samples in centrifuge tubes were prepared on Sept. 17 2014. | # Five samples in centrifuge tubes were prepared on Sept. 17 2014. | ||
#* They were as follows: BSA, lysozyme, [Au]:[BSA]=30:1, [Au]:[Lysozyme]=30:1, and soy protein. Refer to lab manual from last week for detailed information on how they were made. | #* They were as follows: BSA, lysozyme, [Au]:[BSA]=30:1, [Au]:[Lysozyme]=30:1, and soy protein. Refer to lab manual from last week for detailed information on how they were made. | ||
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# Perform electrophoresis | # Perform electrophoresis | ||
# Run for 30 minutes at 200V | # Run for 30 minutes at 200V | ||
===Perform Electrophoresis=== | =====Perform Electrophoresis===== | ||
*Run for 30 minutes at 200V | *Run for 30 minutes at 200V | ||
===Develop/Stain your gel=== | =====Develop/Stain your gel===== | ||
# Place gel in Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes | # Place gel in Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes. | ||
#*Note: our gel was placed on top of Paul group's gel | |||
# Place gel in Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 1 hour | # Place gel in Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 1 hour | ||
#*Note: our gel was placed on the bottom of the container | |||
# Place gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes | # Place gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes | ||
#* Repeat this step with fresh destain solution 2 more times | #* Repeat this step with fresh destain solution 2 more times | ||
===Bradford analysis for protein content=== | |||
*Both for dialyzed and non-dyalized BSA colloid solutions | |||
*Also for outside solutions | |||
**25000 g/mol MW cut-off glycine soak | |||
**3500 g/mol MW cut-off glycine with colloid | |||
**3500 g/mol MW cut-off H<sub>2</sub>O soak | |||
**3500 g/mol MW cut-off NaCl soak | |||
**3500 g/mol MW cut-off glycine soak | |||
Note: protein seemed to hsve precipitated out of solution <br.> | |||
Bradford analysis of outside solutions: | |||
#600μL of outside solution | |||
#200μL Bradford reagent | |||
# 200μL Tris/NaCl buffer | |||
Run UV-Vis from 400 to 800 nm. |
Revision as of 12:34, 23 September 2014
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Task for September 2014
Procedure followed as described by Dr. Hartings on Sept. 25 2013 ElectrophoresisPrepare the Gel and Assemble the Electrophoresis Cell
Note: 200mL of running buffer (10x SDS-PAGE running buffer 30.3 g Tris, 144.1g Clycine, 10g SDS, water to 1L) were diluted to 2 L. This amount was necessary in order to fill up the inner and outer buffer chambers Prepare and Load Samples
Perform Electrophoresis
Develop/Stain your gel
Bradford analysis for protein content
Note: protein seemed to hsve precipitated out of solution <br.> Bradford analysis of outside solutions:
Run UV-Vis from 400 to 800 nm. |