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. >

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*

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*

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.

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 
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 
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

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
Calculation errors
Length of the experiment

Tuesday, February 15, 2011

Zip Tip Protocol~ C18 tips

The zip tip protocol is used to clean up the biological samples after protein digestion.  Its main purpose is to get rid of the chemicals used in protein digestion, such as DTT, IAA, salts, SDS, etc.

Three solutions need to be prepared in order to proceed with the procedure: solution A, solution B, and solution C.

Solution A: Rinse Solution (0.1% formic acid 100%)
Final volume:1000 ul
(100%)V=(0.1%)(1000 ul)
V=1 ul FA 100%
*Obtain 1 ul FA 100%, 999 ul of water*

Solution B: Wet Solution (0.1% FA 100%, 50% acetonitrite)
Final Volume: 1000 ul
*Obtain 1 ul FA 100%, 500 ul ACN, 499 ul water*

Solution C: Elution Solution (0.1% FA 100%, 60% ACN)
Final volume: 1000 ul
*Obtain 1 ul FA 100%, 600 ul ACN, 399 ul water*

(Added 70 ul of water to the dried biological samples)
1. Set pipette to 10 ul.
2. Equilibrate tip with wet solution B.  Aspirate wet solution into the tip.  Dispense to waste.  Repeat twice.
3. Aspirate rinse solution A.  Dispense to waste.  Repeat once.
4. Bind peptides.  Aspirate and dispense the sample 10 to 20 times (depends on sample complexity).
5. Aspirate rinse solution A and dispense to waste.  Repeat 7 to 10 times.
6. Elute peptides.  Set pipette to 5 ul.  Aspirate 5 ul of elution solution C into the tip and place in a clean vial.  Repeat once or twice.
Dilute to desired final concentration with spraying solution: 0.1% FA, 50% ACN (never actually made this because for my lab it is unnecessary, my mentor and I just dried up the samples again after these steps)

Monday, January 24, 2011

Trypsin Digestion Protocol~

 Proteins are digested and chopped up into little pieces for further experiments.  In order to digest proteins, three solutions are used: DTT, IAA, and NH4HCO3.  A type of enzyme will be used to cut the protein at a specific amino acid.


1. DDT (Dithiothreitol) breaks up disulfide bonds formed by the bonds of cysteine. So, DTT is used to "unfold" the proteins and eliminate the bonds that make the protein folds.
  Prepare 1 ml of 100 ml of DTT
    molar weight: 154.25 g/m
     100 mM in 1000 ml= x mM in 1 ml
      x= 0.1 mM= 1*10^-4 M
      x=0.01542 g =15.42 mg
2. IAA (Iodoacetamids) stabilizes the disulfide bonds broken up by DTT.  IAA prohibits the disulfide bonds to bond again.

  Prepare 1 ml of 100 ml of IAA
    molar weight: 184.96 g/m
     100 mM in 1000 ml= x mM in 1 ml
      x= 0.1 mM= 1*10^-4 M
      x=0.018496 g =18.496 mg
3. NH4HCO3 is a buffer that stabilizes and helps the enzyme to rise to its maximum capability to clip and cut the protein.  A certain enzyme functions better with certain pH.  In this case, NH4HCO3 has a pH that works best with trypsin.
  Prepare 1 ml of 100 ml of NH4HCO3
    molar weight: 79.06 g/m
     100 mM in 1000 ml= x mM in 1 ml
      x= 0.1 mM= 1*10^-4 M
      x=0.007906 g =7.906 mg

Two proteins used: BSA and apomyoglobin
1. BSA contains disulfide bonds; thus DTT and IAA are added to break the bonds.
  10 ul of BSA (3 ug/ul)given, add:
     5 ul DTT,
     25 ul NH4HCO3, and
     20 ul water.
     >incubate at 50 C for 45 minutes

    25 ul IAA, and
    25 ul NH4HCO3.
    >sit in darkness for 1 hour

The enzyme is then added according to a protein to enzyme ratio.  In this case, I used 50:1 (protein to enzyme in ug)
  BSA given: 10 ul of 3ug/ul
  50:1 = (3 ug/ul)*10 ul: x
  x= 0.6 ug
   trypsin stock concentration: 0.1 ug/ul
   0.1 ug/ul*x= 0.6 ug
   x=6 ul

   >add 6 ul of trypsin in the BSA solution and incubate at 37 C for 10 hours

2. Apomyoglobin only undergoes the protein digestion process
  Apomyoglobin given: 20 ul of 1 ug/ul, add:
    2 ul trypsin, and
    22 ul NH4HCO3.
   > incubate at 37 C for 10 hours

Sunday, January 16, 2011

Bradford Protein Assay~

Happy LATE New Years~ I am sorry, I been busy studying for school (finals is coming up) and organizing data from the lab into my notebook. However.....

Recently, in my lab at CalTech, I did a lab on Bradford protein assay.  It is a procedure based on an absorbance shift of the Coomassie Brilliant Blue G250 dye.  When the dye binds with proteins, it convert from its original red form into a bluer form.  The bound form of the dye has an absorption spectrum maximum held at 595 nm.  Certain concentration of detergents, which is used to lyse cells, interferes with the Bradford assay.

I used Bovine Serum Albumin (BSA) as a known protein to create a calibration graph of concentration versus absorbance.

Prepare BSA solutions:

I was given BSA solution of 1 ug/ml, and I need to prepare BSA concentrates at 2 ug/ml, 5 ug/ml, 7 ug/ml, 10 ug/ml, 20 ug/ml, and 30 ug/ml.

Concentration (ug/ml)
BSA solution (based on 1 ug/ml)
Bradford reagent dye
2 ul
998 ul
5 ul
995 ul
7 ul
993 ul
10 ul
990 ul
20 ul
980 ul
30 ul
970 ul

All of the solutions have the final volume of 1000 ul or 1 ml.

I, then, input 2 ul of the solution into the nanometer, which has the Bradford protein assay procedure pre installed into the computer.

^ Instrument I used.

^The display.

A calibration graph is formed on the concentration versus absorbance.  The graph uses the Beer-Lambert Law:
(where Eb are constant, sometimes written as k and c as the concentration)

The calibration graph look something like this:

Notice the r squared variable.  In statistics, it is known as the coefficient of determination.  It determines the linearity of the graph.  In other words, the closer the variable is to 1, the more linear it is.  

Using the calibration graph, I was able to determine the concentration of several biological samples based on the absorbance of the samples and the corresponding absorbance over concentration.  

The following are a list of biological samples, which I performed the Bradford assay on in order to find the concentration:

Possible sources of error:
I performed the calibration graph and the actual experiment on two different days, so the results are not as accurate as they possibly are.
There are some samples with 0 concentration.  One thing is that I did not sanitize the nanometer correctly or air went into the machine while the experiment is performed.

This conclude the experiment. XD

Wednesday, December 15, 2010

Introduction to....well....Research

Hi, welcome to my blog!!! Glad you can make it here. (it took me 30 long minutes T_T)  But I am quite excited to be here, I am really interested in science and hopefully become a participates in the medical field.  Because I joined a program at school, I am forced (correction: willingly) to find a lab.
And yea...I just started research here at Caltech several weeks ago, not knowing anything about anything, except the basic chemistry course I took at my high school (yea, I am still in high school).  When I first came to the lab, I was astonished by how much instruments and machines it has in it.  I probably don't know how to use 99% of it.  My mentor was nice enough to teach me stuff from the beginning...starting with....the pH meter.  Since my high school is too poor to even get pH meters, I only read about it in the book.  Then again, I read a lot of things in books, but I never get to try any of those.  I guess this can be my chance trying to learn stuff I never learn in school, and get experience from researches I conduct here.
Thank you for viewing my blog, ciao ciao. :)