Protein in Pig heart

Day 1 search articles about purification of mMDH from pig heart.
mMDH is known for participating in the tricarboxylic acid cycle, 
mMDH is an isoform of MDH.
N-term: MLSALARPAG
C-term: EEFVKNMK( 20 amino acids)
Follow the method: The purification of mMDH from bovine heart
First step: define the protein that includes N and C-Term
Search on internet to find out the protein
To find the name of the protein: type the  ´´ MLSALARPAG´´ at this link
mMDH( mitochondrial malate dehydrogenase) is from Heart pig.
- Decide which method going to be used
The purification process is performed following steps:
- Choose buffer
- the protein will be separated chromatographically: anion- exchange and affinitiy( to enhance the purity)
- the reasons for choosing anion-exchange chromatography are:
the column: DEAE-Sepharose CL6B
- the buffer going to be used is Tris(HCl)
the 2nd column is Blue Sepharose CL-6B following the method in this article
Day 2
1. the preparation of pig heart
- Prepare potassium phosphate buffer pH = 7, 50mM
- 6,8g KH2PO4 + 50ml H20----> 1M KH2PO4
- 8,7 g K2HPO4 + 50 ml H2O -----> 1M K2HPO4
Dilute 20 times to have 50mM each of solution
Monitor pH by pH meter, Mix these solution, pour KH2PO4 solution into K2HPO4 and  until obtaining pH = 7.
Ratio 18(K2HPO4)/7(KH2PO4) = 2,57
- Pig hear stored at 4 degree C, is cut into small peaces, take 15g then but into 30 ml buffer(50mM) containing MeSH(0,2% v/v = 60 ul). The use of MeSH is to ''break'' the bond S-S from protein, the tertiary structure and the quaternary structure of some proteins(including mMDH) can be disrupted.
- Centrifugate the sample in 30 min with speed 11000
 Day 3:
Test enzyme activity of mMDH based on the oxidation of
L-Malate + NAD+ <(2)------(mMDH catalyst)-----(1)> beta-NADH +  oxaloacetate + H+
L-Malate is a substrate 
- beta-NADH deduces the changes of absorbance. In this case, The absorbance will negatively increase.
- Enzyme activity = moles of substrate converted per unit time = rate × reaction volume
Be aware of that oxaloacetate is not stable so the direction (1) should be taken into account.
- Prepare phosphate buffer, and the subtrates following:
* sample 1: 0,2ml-10mM NAD+ 0,2ml-10mM L-Malate + 0,1 opened cells pig heart and 2,5ml buffer phosphate = total 3ml
* sample 2:  0,2ml-20mM NAD+ 0,2ml-20mM L-Malate + 0,1 opened cells pig heart and 2,5ml buffer phosphate = total 3ml
* sample 3:  0,2ml-30mM NAD+ 0,2ml-30mM L-Malate + 0,1 opened cells pig heart and 2,5ml buffer phosphate = total 3ml
* sample 4:  0,2ml-40mM NAD+ 0,2ml-40mM L-Malate + 0,1 opened cells pig heart and 2,5ml buffer phosphate = total 3ml.
Run the spectrometer at 340nm wavelength, running time 1 min.
The protein concentration can be determined by the Bradford following protocol
Note: the cuvet must be up and down after the sample is placed in, the enzyme activity is detected only when the cells  are opened meaning that the protein in this case mMDH is extracted from the cells.
The results: The Absorbance from the spectrometer provides no information about the enzyme acitivity, the reasons the the protein was not extracted from the cell or the pH is so low that ''kill''the protein.
The temperature does not affect the detection of enzymes. After about 30 seconds, the reaction reaches equilibrium, the absorbance is not changed any more.
    The pig heart is stored at -20 degree C, then cut into small pieces that are going to be blended. Because the solid pieces are blended, the cells will be opened effectively.
   c-MDH, and LDH will be in these fractions
- The activity showed at highest level when the concentrations  NAD+, L-malate are 40mM.
Day 4: Run chromatography
Change the buffer Tris pH = 8
 - Extract (open cells) pig heart again, but after blending,
- Perform the enzyme activity, the result obtained is very similar to ones in Day 3.
- The enzyme activity was found.
Day 5: Run Chromatography
Read guideline before using.
Following steps: 
1. Wash the column, pump, tube and the whole chromatography system, by ethanol
2. The eluent will be 20mM Tris(HCl)/pH 7.0, MeSH 0.2%(v/v) prepared 1000 mL.
3. For the elution of Protein, at least 500ml of Tris(HCl)/pH 7.0, MeSH 0.2%(v/v) 100mM KCl was prepared.
4. The sample is diluted 5 times. Inject the sample to the injection port(injection loop volume: 2ml), keep the syringe in the injection port. Set flow rate 0.5 ml/min, collection fraction: 1ml, and gradient mixture.

- The results: From the UV-Vis detector, and chromatogram obtained from the computer, No infor about enzymes is noticed. 20 fractions collected ----> throw away!!!!
The reasons: What happened if 2 buffer solution Potassium phosphate and Tris were mixed together??? Because the buffer for extraction of protein is Potassium phosphate differentiates from the Tris/HCl buffer.
The next day, the Tris will be used as buffer for extraction of protein?? Should be??
The unbound frations will be collected containing mMDH. c-MDH, and LDH will be in these fractions bounding to the column and will be eluted.

Day 6
- Extract mMDH again, the buffer pH =7
- Prepare potassium phosphate buffer pH = 7,1M
- 27,2g KH2PO4 + 200ml H20----> 1M KH2PO4
- 34,8 g K2HPO4 + 200ml H2O -----> 1M K2HPO4
Day 7
- Prepare 100mM  NAD+ (M= 721.44), 100mM  L-malate(M=156.1) in phosphate buffer pH=7
- Continue to run anion exchange chromatography, with column: DEAE-Sepharose CL6B (15 ml,
2.2X4 cm ID) and from the chromatogram, the fractions are collected and tested for enzyme activity(Day 3)

-  Protocols to make SDS-PAGE gel
Resolving Gel
                                         12%(10ml)                     14%(10ml)
                    
30%  Acryl-Bis:            4.0ml                                 3.5ml
1.5M Tris/HCl, pH=8 : 2.5ml                                 2.5ml
10%SDS                         100ul                                 100ul
Distilled water                3.3ml                                3.8ml
10%APS                         100ul                                 100ul
TEMED                           5ul                                      5ul

Stacking Gel                    4%(10ml)
30% Acryl-Bis                 1ml
0.5M Tris/HCl pH=6.8    2.5ml
10%SDS                           100ml
Distilled water                  6ml
10%APS                            100ul
TEMED                              10ul
Running Buffer
10x
Tris                    30g/L
Glycine               144g/L
SDS                   10g/L
To make the running buffer, the 10X is diluted 10times, the 10X buffer is made for convinience.
4X sample buffer
Tris, pH=8, 250mM
Glycerol     40%(v/v)
the sample is diluted 4 time, then is taken to further treatment
SDS(powder)   9.2%(w/v)
Beta-Mercaptoethanol 20%(v/v)
Bromophenol blue    Trace of 2mL of a solution of 0.1%(Bromophenol is insolutable in water)
Staining solution
1 tablet of Coomassie in 1.0L of 0.5%(v/v) acetic acid.
De-staining solution
30% acetic acid

The sample(30ul+30ul sample buffer) must be boiled for 5 min at 95 degree C in 1Xsample buffer.
Centrifugate in 10min, After centrifugation the sample is taken to other container.
Make the SDS-PAGE
- After all the solutions prepared. They are mixed together, 10%APS and TEMED is put in the solution in the last, because they cause the polimization. After filling the Resolving Mixed solution, the water is poured above. Wait until the soluion turns into Gel(polimized). Add Stacking Gel.
Day 8
Run Electrophoresis
The fractions 25-32 collected are mixed with 1X sample buffer respectively, boiled at 95 degree C, centrifugated.
Take 10uL, fill in SDS-PAGE.
Then put in the 10X running buffer, run in 1hour.
Day 9
- Check the SDS-PAGE to find the protein,
- Making extract pig Heart.'
Day 10
The unbound fractions containing mMDH is tested emzyme activity. And then run with Blue Sepharose CL-6B column.
- Tris/HCl pH = 7.6
Day 11 Presentation
Day 12 Continue with purification of unbound fractions by affinity chromatography.
And cation exchange chromatography but other contaminants or proteins can be subjected to cation exchange column because they will be retained by the column.
Day 15 
Note: - The protein must be qualified in order to calculate how much the protein is purified at the starting point after extraction to the final point after running affinity chromatography.
- Some groups on the protein also are responsible for the absorption UV-Vis at 280 nm,this is the sample way to test enzyme activity
- Chose the right column, for the affinity column, decide and find which groups on the column will attract or retain the protein, pH, and buffer..