User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/18: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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== | ==SDS-PAGE== | ||
#Our protein solution was collected from the cold room | |||
#It was filtered using filtration apparatus | |||
#It was transferred to a 50mL falcon tube labeled Purified Hb, DML 7/18/2014 | |||
#We will run a gel with the solution and past solution to check purity | |||
#The gel will be set up in the following order | |||
##Myoglobin and BSA ladder | |||
##Empty | |||
##Purified protein solution | |||
##1/10 dilution of Purified protein solution | |||
##Empty | |||
##Ammonium sulfate treatment pellet dissolved in 50mM Tris | |||
##1/10 dilution of Ammonium sulfate treatment pellet dissolved in 50mM Tris | |||
##Empty | |||
##Cell pellet dissolved in 50mM Tris | |||
##1/10 dilution of cell pellet dissolved in 50mM Tris | |||
##Empty | |||
##Myoglobin and BSA ladder | |||
==Gel Electrophoresis== | |||
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 | |||
## Load 18uL of ladder and sample in the wells | |||
# 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 for 30 minutes | |||
## Place gel in Stain Solution for 1 hour | |||
## Place gel in Destain Solution for 15 minutes | |||
### Repeat this step with fresh destain solution 1 more time | |||
==Stock Solutions== | |||
#Protein ladder | |||
-1mg BSA and 1mg Myoglobin in 1mL water | |||
-Solution was diluted to 1/10 to be used in gel | |||
#1X Running Buffer | |||
-14.4g Glycine, 1g SDS, 3g Tris in 100mL H2O | |||
-Solution was diluted to 1L with water | |||
#Fixative Solution | |||
-40 Methanol, 10% Acetic Acid, 50% Water | |||
#Stain Solution | |||
-90% water, 10$ Acetic Acid and .0025% Commassie Brilliant Blue | |||
#Destain Solution | |||
-90% Water and 10% Acetic Acid | |||
==Results== | |||
[[Image:Gel 71814 DML.jpg|500px]] | |||
This gel is oriented so that lane 1 is on the far left. | |||
This procedure did not purify the protein as expected | |||
At this point, the most successful method has been the combination of Q column and SP column | |||
Buffers will be prepared for this method | |||
#Prepare buffers | |||
##50mM Tris buffer at pH 8.3 | |||
##10mM Sodium Phosphate buffer at pH 7.2 | |||
#Solutions were filtered using a vacuum filter | |||
#Solutions were placed in the autoclave for 1 hour at 121C | |||
Latest revision as of 00:07, 27 September 2017
Project name | Main project page Previous entry Next entry |
SDS-PAGE
Gel ElectrophoresisWe 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
Stock Solutions
-1mg BSA and 1mg Myoglobin in 1mL water -Solution was diluted to 1/10 to be used in gel
-14.4g Glycine, 1g SDS, 3g Tris in 100mL H2O -Solution was diluted to 1L with water
-40 Methanol, 10% Acetic Acid, 50% Water
-90% water, 10$ Acetic Acid and .0025% Commassie Brilliant Blue
-90% Water and 10% Acetic Acid ResultsThis gel is oriented so that lane 1 is on the far left. This procedure did not purify the protein as expected At this point, the most successful method has been the combination of Q column and SP column Buffers will be prepared for this method
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