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|style="background-color: #EEE"|[[Image:BDLlogo_notext_lr.png|128px]]<span style="font-size:22px;"> Biomaterials Design Lab</span> | |style="background-color: #EEE"|[[Image:BDLlogo_notext_lr.png|128px]]<span style="font-size:22px;"> Biomaterials Design Lab</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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==Objective== | ==Objective== | ||
* Utilize SDS-Page electrophoresis to observe protein sample solutions | |||
* Redo the Bradford Assay calibration curve with Lysozyme stock | |||
==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 | |||
# The Gel was previously prepared and the Electrophoresis Cell was assembled | |||
## Remove comb and tape from the gels | |||
## Rinse the wells with running buffer | |||
### Added 200mL 10x SDS-Page running buffer to 1800mL of H<sub>2</sub>O | |||
## Assembled the electrophoresis cell (note diagrams in manual) | |||
## Filled the inner and outer buffer chambers with running buffer | |||
# Prepare and Load Samples | |||
## Samples were prepped 09/17 | |||
## Samples were heated for 10 minutes at 100°C in the thermocycler | |||
## Loaded 20uL of protein ladder into column 1 of the gel | |||
## Loaded 20uL of samples into the appropriate lane of the gel | |||
# Performed electrophoresis | |||
## Ran for 30 minutes at 200V | |||
# Developed/Stained the gel | |||
## Placed gel in Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes | |||
## Placed gel in Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 1 hour | |||
## Placed gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes | |||
### Repeat this step with fresh destain solution 2 more times | |||
# Bradford Assay of the Lysozyme Stock | |||
## 0.50350g of Lysozyme was added to 1L of 0.90378% saline solution | |||
## Tris-NaCl buffer prepared with 1.5145g Tris,0.7291g NaCl, and 250mL H<sub>2</sub>O | |||
==Data== | ==Data== | ||
* | * Well Number | ||
** 1 - Protein Ladder | |||
** 4 - BSA Stock Solution | |||
** 6 - Soy Stock Solution | |||
** 8 - Lysozyme Colloid | |||
** 10 - Lysozyme Stock Solution | |||
** 12 - BSA Colloid | |||
*Bradford | |||
{| | |||
| align="center" style="background:#f0f0f0;"|'''Dilution #''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''Initial Lysosyme (μg/mL)''' | |||
| align="center" style="background:#f0f0f0;"|'''Bradford (μL)''' | |||
| align="center" style="background:#f0f0f0;"|'''Bradford (μL)''' | |||
| align="center" style="background:#f0f0f0;"|'''Final Lysozyme (μg/mL)''' | |||
|- | |||
| 1||||503.500||800||200||402.800 | |||
|- | |||
| 2||||251.750||800||200||201.400 | |||
|- | |||
| 3||||125.875||800||200||100.700 | |||
|- | |||
| 4||||62.938||800||200||50.350 | |||
|- | |||
| 5||||31.469||800||200||25.175 | |||
|- | |||
| 6||||15.734||800||200||12.588 | |||
|- | |||
| 7||||7.867||800||200||6.294 | |||
|- | |||
| 8||||3.934||800||200||3.147 | |||
|- | |||
| 9||||1.967||800||200||1.573 | |||
|- | |||
| | |||
|} | |||
The following graphs were produced using UV-Vis data collected analysis of each of the solutions above. It was observed that at concentrations above 20 μg/mL the calibration curve is no longer linear, so the second graph only includes concentrations of lysozyme < 20 μg/mL. | |||
[[Image:Bradford_UV_VIS_Chart.png|500px]] | |||
[[Image:Bradford_Calibration_Curve_9-23.png|500px]] | |||
==Notes== | |||
Bradford Assay is only effective from 1μg/mL to 10μg/mL | |||
[[Category:Course]] | [[Category:Course]] |
Latest revision as of 00:20, 27 September 2017
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Objective
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
Data
The following graphs were produced using UV-Vis data collected analysis of each of the solutions above. It was observed that at concentrations above 20 μg/mL the calibration curve is no longer linear, so the second graph only includes concentrations of lysozyme < 20 μg/mL. NotesBradford Assay is only effective from 1μg/mL to 10μg/mL
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