Biochemistry Lab. Comparative Proteomics
Introduction:
The aim of this experiment was to investigate fish diversity using the technique SDS-PAGE. In
class we performed an SDS-PAGE of fish muscle and were assigned with five different type of
fishes; Red Grouper, Catfish, Shrimp, Red Snapper, Salmon and beef heart. Our table was
assigned to obtain about 0.5 cm of frozen tissue from a shrimp and then we started preparing
the sample for electrophoresis. After this was done, we loaded and ran the gels, followed by the
staining process. Finally, by using an evolutionary tree we were able to see the relationship
between each fish.
Purpose: The goal of this experiment is to compare LDH collected from various tissues using
the gel-electrophoresis technique. When electricity flows, proteins with a negative charge are
pushed from the negative pole to the positive pole. Smaller molecules flow more quickly across
the gel, whereas bigger ones move more slowly.
Introduction: Lactate dehydrogenase (LDH) is a metabolic enzyme that catalyzes the
conversion of pyruvate to NADH/NAD (+). LDH is an enzyme present in practically every cell in
our body, including the blood, muscles, brain, kidneys, and pancreas. When cells are wounded
or destroyed, LDH, a tetrameric enzyme composed of two main subunits, LDHA and LDHB, may
assemble into five different iscenzymes: H4, MH3, M2H, M3H, and M4.
LDH (lactic dehydrogenase) has a molecular weight of 140 KDA and is composed of four
subunits: M (muscle) and H (heart) (heart). LDH has molecular weights of 35, 70, and 80 KDA in
monomer, dimer, and tetramer forms, respectively. Using agarose gel electrophoretic to
numerous LDH isozymes, we will identify LDH isozymes based on their electrophoretic
migrations, estimate their molecular weight (MW), and explore the charge nature of the H and M
subunits.
-What is LDH?
Lactate dehydrogenase is an enzyme found in nearly all living cells. LDH catalyzes the
conversion of lactate to pyruvate and back, as it converts NAD⁺ to NADH and back. A
dehydrogenase is an enzyme that transfers a hydride from one molecule to another. LDH exists
in four distinct enzyme classes.
-Which is the LDH structure?
Human LDH is a quaternary protein formed of the combination of two subunits, M and H
(Muscle and Heart) into a structure of four of the subunits.
-Which is the molecular weight of LDH monomer and the tetramer?
The LDHA molecular weight is 35 kDa, 70 kDa and 140 kDa for monomer, dimer and tetramer
forms,
-Define the concept of isozyme?
any of the genetically variant forms of certain enzymes that catalyze the same reaction but that
may differ in activity, composition, or physical properties
-Which are the LDH isoenzymes subunits?
Two distinct subunits combine to form the five tetrameric isoenzymes of lactate dehydrogenase.
The LDH-5 subunit (muscle type) has higher maximal velocity (Vmax) and is present in
glycolytic tissues, favoring the formation of lactate from pyruvate.
-Where are the LDH isoenzymes found?
LDH isoenzymes are found in many tissues in the body, including the heart, red blood cells,
liver, kidneys, brain, lungs, and skeletal muscles.
Materials:
●
●
●
●
●
●
●
●
●
●
●
●
Laemmli sample buffer, 30ml.
Precision Plus Protein Kaleidoscope prestained standards, 50 μL.
10x Tris-glycine-SDS electrophoresis buffer, 1L.
Bio-Safe Coomassie stain for protein, 100 ml.
Actin and myosin standard, 500 µg lyophilized.
Dithiothreitol, 0.3g.
Prot/ Elect pipet tips for gel loading.
1.5 ml fliptop micro test tube.
1.5 ml screwcap micro test tube.
Disposable plastic transfer pipets.
Gel-staining trays.
Foam micro test tube holders.
●
●
●
●
●
●
●
●
Fish Samples of ( Red Grouper, Catfish, Shrimp, Red snapper, Salmon)
Beef heart LDH
Actin and Myosin samples.
Micropipettes.
Power supply
Water bath.
Vertical gel electrophoresis chamber.
Gel precast polyacrylamide gels, 15%, 10-well each.
Procedure:
1h) Label one 1.5 ml fliptop micro tube for each of five fish samples (it will be one per table).
Also label one screwcap micro tube for each fish sample.
2) Add 250 μl of Bio-Rad Laemmli sample buffer to each labeled fliptop microtube.
3) Cut a piece of each fish muscle (our fish is catfish) about 0.25 x 0.25 x 0.25 cubic cm and
transfer each piece into a labeled fliptop micro test tube. Close the lids.
4) Flick the microtubes 15 times to agitate the tissue in the sample buffer.
5) Incubate for 5 minutes at room temperature.
6) Carefully transfer the buffer by pouring from each fliptop microtube into a labeled screwcap
microtube. We cannot transfer the fish!
7) Heat the fish samples in screwcap microtubes for 5 minutes at 95°C.
8) Set up Mini-PROTEAN Tetra gel box.
9) Prepare a TGX or Ready Gel cassette by cutting along the black line on the bottom of the
cassette with a razor blade and pulling off the plastic strip, as indicated on gel cassette.
10) Remove the comb from the TGX or Ready Gel cassette.
11) Place TGX or Ready Gel cassette into the electrode assembly that has the banana plugs
with the short plate facing inward. Place a buffer dam or another TGX or Ready Gel cassette on
the opposite side of the electrode assembly, with notch on buffer dam facing inward.
12) Push both gels towards each other, making sure that they are against the green gaskets
that are built into the clamping frame; make certain that the short plates sit just below the notch
at the top of the green gasket. Slide the green arms of the clamping frame over the gels, locking
them into place.
13) Lower the electrode assembly with the gels in it into the mini tank on the side of the tank
with the plastic tabs. Make sure that the red banana plug goes on the side of the tank with the
red oval.
14) Completely fill the inner chamber with 1xTGS electrophoresis buffer, making sure the buffer
covers the short plate (~150 ml).
15) Fill mini tank with approximately 700 ml of 1x TGS electrophoresis buffer until the buffer
reaches the 2 gels line on the tank.
16) If using, place sample loading guide on top of the electrode assembly.
17) Heat fish samples and actin and myosin standard to 95°C for 2–5 min.
18) Load your gel.
19) Electrophorese for 30 minutes at 200 V in 1xTGS electrophoresis buffer.
20) After electrophoresis, remove gel from cassette and place two gels in one staining tray.
Rinse gels in tap water. If time allows rinse gels 3 times for 5 minutes.
21) Pour off water and add 50 ml of Bio-Safe Coomassie blue stain. Stain gels for at least 1
hour with gentle shaking for best results.
22) Discard stain and destain gels in a large volume of water overnight, changing the water at
least once. Blue-stained bands will be visible on a clear gel after destaining.
23) Dry gels using GelAir cellophane.
1
2
3
4
5
6
7
8
9
1
0
1
1
1
2
Wells
Empty
Kaleidoscope
Empty
Red Grouper
(Group 1)
Catfish (Group
2)
Shrimp (Group
3)
Red Snapper
(Group 4)
Salmon (Group
5)
Beef Heart
Empty
Actin and Myosin
Empty
Results:
● Select the number of coincident bands between all the tested fish protein samples.
● Take a picture of the gel that will support your observations.
● Mention the MW of the coincident bands taken the caleidoscope bands as reference.
Conclusion:
To determine protein purity and the presence of subunits, Polyacrylamide gel electrophoresis is
commonly used. LDH is broken down into subunits when it is run through an SDS gel. Beef
Herath and skeletal muscle LDH subunits were determined to have molecular weights of
477kda and 387kda, respectively. Because it was difficult to discern the bands based on the
photos, the computations may be wrong. LDH should ideally have no more than 5 bands of
subunits per column. Individual M and H monomers will migrate along the gel instead of a native
tetramer. The primary application of SDS-PAGE is to determine molecular weight. SDS removes
the shape and charge variables, causing all negatively charged proteins to migrate to the
positive end of the gel. There are also several dyes that may be employed during
electrophoresis. Bromophenol blue, which is incorporated in the sample buffer, is the first kind.
This serves as a landmark for us to assess how the separation is progressing. Because
bromophenol blue is negatively charged and tiny, it moves faster than the LDH proteins that
would separate on the gel. The second dye is Coomassie Blue, which is similar to the dye used
in the Bradford protein test. After electrophoresis, it is used to dye all of the proteins blue and
show where they are on the solutions. With the exception of the Liver LDH, which was more
akin to the usual LDH protein molecule, all were highly active and had more bands than I
expected.