Biochemistry Labs Flashcards
6.Paper and Thin Layer Chromatography
Theory;
- Method to measure purity of a compound.
- components are separated between stationary and mobile phase
- Mobile phase: Water adsorbed to a cellulose sheet.
- Stationary phase: Is a developing solution move up the stationary phase.
- Components separate depending on: their ability to absorb to stationary or mobile phase.
- Acetic Acid is used to set the pH.
- Hydrophobic components: - move along the organic front
- Hydrophilic components: - remain near the stationary phase.
Aim: Determine the unknown amino acid in a solution by comparing it’s RF value to known RF Values.
Procedure:
- cut 10x50 cm paper
- mark a line with pencil 6cm from the bottom.
- use a pencil to mark points on the line separated by 1.5cm, and apply solutions with a microcapillary.
- place into chamber, where line is 1cm above the solvent.
- Allow to develop for 2hours then spray with ninhydrine solution.
- Measure RF value.
Thin Layer:
- Similiar to Paper however it takes about 40mins to develop and gives better seperation.
- Involves use of a polyscilinate layer on 0.2mm thickness.
- Water serves as stationary + mobile phase.
Conclusion:
- Proline = yellow colour
- all the others = purple.
- using rf value find out which amino acid it is.
- Reversible Precipitation of Proteins
a)”Salting out” using Ammonium Sulphate.:
Background:
- Globular proteins are soluble in water due to their hydrate envelope.
- When this envelope is disturbed then they precipitate.
- Ammonium Sulphate does not affect it’s secondary or tertiary structure thus this is reversible.
Aim:
To salt out proteins from the Blood Serum with Ammonium Sulphate.
Procedure:
- Add 5ml of Blood serum to a test tube followed by 5ml of Ammonium Sulphate.
- Allow to stand for 10mins (until half saturation point).
- Pour through filter paper - will see Globins on the paper.
- ADD SOLID Ammonium sulphate until saturation, then allow to stand for a few mins.
- Then filter and will see Albumins removed on the paper.
Conclusion:
-Salting out of different proteins occurs at different stages
b)With Organic Solvents
Aim: Show that ethanol causes the precipitation of proteins and that it is reversible.
Theory:
- Ethanol decreases protein solubility.
- It’s polar hydroxyl group interacts with the protein’s groups.
- It’s apolar Hydrocarbon chain is insoluble causing precipitation of proteins.
Procedure:
- Add 5ml of ethanol to 2ml of Blood Serum.
- See precipitation occur.
- Carefully pipette more ethanol on the boundary and see even more precipitation.
- Adding water causes redissolving.
2.Irreversible precipitation of Proteins
a) By Heavy Metal Salts
Aim: -To show effect of Heavy metal salts on protein
-effect of EDTA on the complex.
Background:
-Ions form covalent co-ordinate bonds with O and N in protein.
-Complex forming ability causes denaturation (irreversibility) and precipitation.
Procedure:
1) put 3ml of Blood serum in 2 test tubes..
2) In test tubes place 1.5ml of lead acetate.
3) in first test tube place 1.5ml EDTA-NA2 and other put 1.5ml of water.
Conclusion:
- Lead acetate causes precipitation, additional drops causes redissolving.
- EDTA prevents complex forming so no precipitation.
b) By Inorganic Acid
Aim: Show effect of inorganic acid on protein solution.
Theory: Inorganic Acids drastically lower the pH which affects the secondary and tertiary structure.
Procedure:
1) Get 3 test tubes.
2) Place 3ml of Blood serum then add drops of HCl/H2SO4/HNO3
3) See that precipitate forms then even more drops causes redissolving.
c)by Heating
Aim: Show precipitation due to heating and influence of various acid/base.
Theory: Heating breaks H-bonds in secondary and tertiary structure.
Procedure:
1) Prepare 5 test tubes.
2) Place Blood serum 2ml in each.
3) 1. Heat only.
2. few drops of 1% acetic acid.
3. 0.5ml of 10% acetic Acid
4 0.5ml of 10% acetic Acid + nacl
5. 0.5ml of 10% NaOH
Conclusion:
1: precipitate form
2. Precipitate forms because pH close to Isoelectric pH>
3: No precipitate as pH too low
4. Precipitate forms due to treatment with salts.
5: pH too high so no precipitate forms.
- Gel Filteration
Aim: To seperate proteins based on size differences.
Procedure note: talk procedure before theory in this one:
1) In this experiment we used a Sephadex 25 Column.
2) We placed 0.3ml of the Bovine Serum/Potassium iodide mix on top of the gel.
3) We then add water as the elution buffer.
4) collect twenty 2ml samples in tubes.
5) after collection add: 1ml SULFOSALACILIC which causes protein precipitation
6) then add 0.5ml of NaOCl (turns iodide brown thus allowing detection).
Theory + Results:
- Gel filteration seperates proteins based on the size differences.
- The gel has pores with a certain diameter.
- In our experiment the iodide is able to enter the Pores thus distributed between pores and amongst the beads.
- Whereas proteins too big to enter to pass between the beads thus move through the column much quicker so are seen in first samples.
Important Formulas:
1.
(ClO-) + (2H+) + 2I-) —-> I2 + Cl- + H20
2.
I2 + I- I3- yellow colour
3.
n-I3 + starch –> Dark blue colour.
10.Determination of Urease Enzyme Kinetics.
Aim:
-To determine the Vmax and Km of Urease.
Theory:
-Urease is an enzyme which catalyses the hydrolysis of Urea into Ammonia and CO2.
EXTRACTION
-We extract it from crushed soybean with a 3:1 Water to Acetone mixture. Also EDTA + 2-Mercaptoethanol is in the micture.
BERTHELOT
- We follow the Urease Activity by using the Berthelot Reaction.
- The Berthelot Reagent is an Alkaline solution consisting of Phenol and Hypochlorite.
- it reacts with Ammonia to form a BLUE-coloured product
There is a direct proportionality with between product formed and time of incubation at all concentrations of substrate/enzyme.
We find that [S} (x-axis) and V (y-axis) forms a hyperbolic curve.
Procedure:
- Prepare 9 Test-tubes.
- 9th Test tube = blank, has 1ml 1% EDTA.
- Remaining test tubes also have 1ml 1% EDTA.
1,2: 50ul of 5mM Uurea
3,4: 50uL of 10mM urea
5,6: 50uL of 20mM urea
7,8: 50ul of 30mM Urea
- Measure Absorbance vs the blank tube.
- Calculate velocity;=Ab/Min
- Plot Line-weaver Burk Plot.
FIND KM AND Vmax
- Reversible and Irreversible Inhibition of Urease Activity.
Aim:
To examine the properties of Competitive and Non-Competitive Inhibitors
Theory:
A ….
->Irreversible Inhibitor: dissociates slowly from enzye as it is tightly bound.
->Reversible inhibitor: dissociates rapidly from the enzyme as it is loosely bound.
->Competitive Inhibitor:
These bind to the active site of an enzyme, thus preventing substrate from binding. They affect the Km.
Their degree of inhibition depends on:
- Binding constant
- Substrate/Enzyme Concentration.
->Non Competitive Inhibitor:
These bind to region apart from the active site. They affect Vmax. They prevent conversion of substrate into product.
In our Experiment:
- Guanidine = Competitive inhibitor
- PCMB = Non-Competitve Inhibitor
Procedure:
Test Tubes are prepared,
1,2: Blanks, 10mM urea + UREASE
Competitive: 3,4: 10mM Urea + Urease + Guanidine 5,6: 20mM Urea + Urease + Guanidine Non-Competitive: 7,8: 10mM Urea + Urease + PCMB 9,10: 20mM Urea + Urease + PCMB 11,12: 10mM Urea + Urease + PCMB + 2-Mercaptoethanol.
- Principle of Denaturing Polyacrimide Gel Electrophoresis of Proteins.
Aim:
-To seperate Proteins based on the Molecular Mass of the Proteins.
Theory:
- In Gel Electrophoresis proteins are seperated by their size.
- We prepare the Sample by boiling the protein mixture with Bromophenol, Glycerol and SDS.
- The FUNCTION OF SDS:
- >It keeps the insoluble denaturised Proteins soluble by binding to the surface.
- > It equalises the negative charge of the polypeptide chain thus separation is only determined by Mr.
-Smaller molecules experience less resistance thus travel greater distances in the gel.
- THE GEL in our experiment consists of two parts:
- > Stacking Gel (pH 6.8)
- > Lower Gel (pH 8.8)
Procedure:
1) Pour Running Gel in the Outer and Inner Chamber
2) Remove the Comb and apply the protein sample with a syringe.
3) Then apply Bromophenol into the well (this is a negative charged dye which allows us to visibly follow the process.
4) Connect the apparatus to a Power Supply and allow separation until blue line reaches the end of the Gel.
5) Apply Commaise Blue to make Protein Bands visible.
