Tuesday, May 14, 2013

Protein Gel Electrophoresis~Colloidal Blue Staining Kit from Invitrogen

The gel is used to observe the relations between the masses of different proteins and to select pieces of protein gel for in gel digestion.
This the machine I will be using for the experiment. >

PREPARATION OF BUFFERS
The two types of buffers are needed to run protein gel: SDS running buffer and ammonium sulfate.

SDS Running Buffer: used to cool down the gel running process
original buffer: SDS running buffer 20x
final buffer solution: SDS running buffer 1x in 1000 ml
20x*V=1x*1000 ml
V=50 ml
*Obtain 50 ml of SDS running buffer and 950 ml of water*

Ammonium sulfate: used for gel storage
Prepare 20% w/v ammonium sulfate in 40 ml
20%=w/40 ml
w= 8 mg
*Obtain 8 mg of ammonium sulfate and 40 ml of water*

PREPARATION OF PROTEIN GEL SOLUTIONS
Proteins must be prepared to be able to run in the gel.  The sample buffer contains SDS, dye, and glycerol.  5 ul of sample buffer is added to the biological samples.  Mercaptoethanol disrupts the disulfide bonds in the proteins.  0.2 ul of mercaptoethanol is added to each biological sample.

BSA (stock solution: 1ug/ul) protein gel: *Obtain 14.8 ul of BSA 1ug/ul, 5 ul of the sample buffer, 0.2 ul of mercaptoethanol 99% v/v*

Ubiqutin (stock solution: 1mg/ml) protein gel: *Obtain 14.8 ul of ubiqutin 1mg/ml, 5 ul of the sample buffer, 0.2 ul of mercaptoethanol 99% v/v*

Ribonuclease A (stock solution: 0.82 mg/ml) protein gel: *Obtain 14.8 ul of Ribonuclease A 0.82 mg/ml, 5 ul of the sample buffer, 0.2 ul of mercaptoethanol 99% v/v* ; *Obtain 10 ul of Ribonuclease A 0.82 mg/ml, 5 ul of the sample buffer, 0.2 ul of mercaptoethanol 99% v/v, 4.8 ul of water*

Carbonic Anhydrase (stock solution: 1.74 mg/ml) protein gel: *Obtain 8.6 ul of Carbonic Anhydrase 1.74 mg/ml, 5 ul of the sample buffer, 0.2 ul of mercaptoethanol 99% v/v, 6.2 ul of water*

PROCEDURE FOR NUPAGE BIS TRIS MINI GELS (PROTOCOL)
1. Obtain Bis Tris Mini Gels Plate.
2. Load 200 ml 1x SDS running buffer into the upper (inner) buffer chamber and 600 ml 1x running buffer into the lower (outer) buffer chamber.
3. Inject protein marker 5 ul into the 2nd well.
4. Insert 20 ul of protein gel solution for each well.
5. Run the gel for 35 minutes.
6. Take out the gel plate and discard the plate.  Keep gel.
 ^ How the gel looks after it was taken from the mini gel, without staining.



STAINING NUPAGE NOVEX BIS TRIS GELS
The gel needs to be stained in order to see the proteins in the gel.  Two solutions need to be prepared to stain the gel: fixing solution and staining solution.


Prepare the Fixing Solution as described in the table below. For best results, prepare the solution fresh prior to staining. 

Fixing Solution
1 Gel
2 Gels
3 Gels
4 Gels 
Deionized Water
40 ml
80 ml
120 ml
160 ml 
Methanol
50 ml
100 ml
150 ml
200 ml 
Acetic Acid
10 ml
20 ml
30 ml
40 ml 



Be sure to shake Stainer B solution before using.  Prepare the Staining Solution as described below without Stainer B.  


Staining Solution
1 Gel
2 Gels
3 Gels
4 Gels 
Deionized Water
55 ml
110 ml
165 ml
220 ml 
Methanol
20 ml
40 ml
60 ml
80 ml 
Stainer A
20 ml
40 ml
60 ml
80 ml 
Stainer B
5 ml
10 ml
15 ml
20 ml



Procedure
1. Shake the gel in the Fixing Solution for 10 minutes at room temperature.
2. Shake the gel in the Staining Solution without Stainer B for 10 minutes at room temperature.
3. Add Stainer B to the existing Staining Solution in the proper volume.
4. Shake gel in Staining Solution for a minimum of 3 hours and a maximum of 12 hours.
5. Decant Staining Solution and replace with 200 ml of deionized water per gel. Shake gel in water for at least 7 hours. Gel will have a clear background after 7 hours in water. Note: Gels can be left in water for up to 3 days without significant change in band intensity and background clarity.
6. For long-term storage (over 3 days), keep the gel in 20% ammonium sulfate solution at 4°C.

^ The outcome of the gel after staining.  The first one visible is the protein marker.  The second column is histone.  The next two is my RibA, they are consistent in mass and is relevantly light.  The heaviest one is BSA.  It has two bands indicating there are two type of proteins in the sample.  The last one and the third to last one are the same, consistent and contain 3 types of proteins.  The second to last one in the columns is my ubiqutin.  It is the lightest in mass compared to all the other samples.  
**********************************************************************************
My mentor and I cut the protein gels in the band that we think are important for further experimenting.

Possible sources of error:
Pipetting errors
Contamination
Calculation errors
Length of the experiment

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