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 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 H2O
- 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 H2O
Data
- Well Number
- 1 - Protein Ladder
- 4 - BSA Stock Solution
- 6 - Soy Stock Solution
- 8 - Lysozyme Colloid
- 10 - Lysozyme Stock Solution
- 12 - BSA Colloid
Dilution #
|
'
|
Initial Lysosyme (μg/mL)
|
Bradford (μL)
|
Bradford (μL)
|
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.
Notes
Bradford Assay is only effective from 1μg/mL to 10μg/mL
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