User:Zachary I. Mendel/Notebook/Zacks Notebook/2013/09/25: Difference between revisions

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==Entry title==
==Objective==
* Insert content here...
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
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|}
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==Notes==
Table 1: Samples in Wells
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Well Numbers'''
| align="center" style="background:#f0f0f0;"|'''Sample'''
|-
| 1||Hemoglobin
|-
| 2||Pepsin
|-
| 3||Pepstatin
|-
| 4||0.5hrs Pepsin
|-
| 5||0.5 hrs Pepstatin
|-
| 6||1 hr Pepsin
|-
| 7||1 hr Pepstatin
|-
| 8||1.5hrs Pepsin
|-
| 9||1.5 hrs Pepstatin
|-
| 10||2hrs Pepsin
|-
| 11||2hrs Pepstatin
|-
| 12||Hemoglobin
|-
|
|}
SDS-Page Gel
[[Image:FLUBBER.jpg|750px]]
Absorbance Spectrum of Pepsin with Hemoglobin
<br>
[[Image:Hemopepsin09251013zem.png|750px]]
Corrected Absorbance Spectra of 20μM Pepstatin with Hemoglobin
[[Image:Pepstathemogl09252013zem.png]]
For our gel we placed it in 10 mls fixing solution. We then dumped out the fixing solution and stained it with 10mls of a staining solution. This stayed on for 1 hour
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Revision as of 16:33, 1 December 2013

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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 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

  1. Prepare the Gel and Assemble the Electrophoresis Cell
    1. Remove comb and tape from the gels
    2. Rinse the wells with running buffer
    3. Assemble the electrophoresis cell (note diagrams in manual)
    4. Fill the inner and outer buffer chambers with running buffer
  2. Prepare and Load Samples
    1. You prepped your samples yesterday
    2. Heat your samples for 5 minutes at 100C (in the thermocycler)
    3. Load 20uL of protein ladder into column 1 of your gel
    4. Load 20uL of your samples into the appropriate lane of your gel
  3. Perform electrophoresis
    1. Run for 30 minutes at 200V (I need to make sure our power source can do this)
  4. Develop/Stain your gel
    1. Place gel in Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes
    2. Place gel in Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 1 hour
    3. Place gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes
      1. Repeat this step with fresh destain solution 2 more times

Notes

Table 1: Samples in Wells

Well Numbers Sample
1 Hemoglobin
2 Pepsin
3 Pepstatin
4 0.5hrs Pepsin
5 0.5 hrs Pepstatin
6 1 hr Pepsin
7 1 hr Pepstatin
8 1.5hrs Pepsin
9 1.5 hrs Pepstatin
10 2hrs Pepsin
11 2hrs Pepstatin
12 Hemoglobin

SDS-Page Gel


Absorbance Spectrum of Pepsin with Hemoglobin


Corrected Absorbance Spectra of 20μM Pepstatin with Hemoglobin

For our gel we placed it in 10 mls fixing solution. We then dumped out the fixing solution and stained it with 10mls of a staining solution. This stayed on for 1 hour

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