Practicals Flashcards
What is an assay
An indirect method for quantifying a substance or activity of biochemical interest
Why is it known as the Bradford Assay
It uses the dye Coomassie Brilliant Blue, which was introduced by Marion Bradford
What are the colours associated with Coomassie Brilliant Blue
Green-brown when free in acid solution
Becomes blue when bound to a protein
What does LDH do
How can it’s activity be measured
Catalysed conversion of lactate to pyruvate using NAD as a H+ receptor
By following production of NADH
Which fixes conducting medium is used for electrophoresis of proteins
How is it prepared
A porous medium of aqueous polyacrylamide
Dissolving acrylamide and a cross linking agent
How do you vary the pore size of polyacrylamide gel
Modify the acrylamide monomer concentration during preparation
What happens to proteins in the presence of sodium dodecyl sulphate (SDS)
Proteins are completely unfolded
Why can inherent differences in charge due to amino acid side chains obscured in presence of SDS
Most proteins bind equal amounts of anionic dodecyl sulphate per gram So behave as polyanions with a constant ratio of negative charge to mass
Most proteins bind what amount of anionic dodecyl sulphate per gram
1.4g per gram of protein
Why can multimeric proteins be dissociated into their subunits by SDS
Disulphide bonds have been previously reduced by mercaptoethanol
During electrophoresis with polyacrylamide gel that contains SDS proteins move at speeds determined by what
The size of their SDS-protein complex
What is an isozyme
An enzyme that can exist in more than one molecular form that is usually tissue-specific
AKA isoenzyme
How many isoenzymes exist of LDH
Why
Five
because it has four sub units and can be encoded by 2 different genes (A and B) so 5 combinations of A and B are possible
ie. A4, A3B1, A2B2, A1B3, B4
How would you find the origin of an LDH isoenzyme in a diseased serum
Compare the activity forms in the gel with that of the LDH from a variety of rat tissue extract by subjecting them to non-denaturing PAGE
Stain the gel with a substrate mixture that turns purple in regions where LDH is found
Why is the Bradford assay not appropriate for diagnosis of a mild heart attack
It will not give specific results for the specific isozyme of LDH in the serum
What is an antibody comprised of
They are white shaped molecules, comprising four chains, two light and 2 heavy
Describe the light and heavy chain components of an antibody
Light: Contains one part of Ig domains
Heavy: Contains two pairs of Ig domains connected by a flexible linker
How are the light and heavy chains of an antibody linked together
By a disulphide bond
True or false:
Each arm of an antibody will bind to a different antigenic component
False
both find the same antigenic component
What produces antibodies
When are the antigen binding sites produced
β cells
During β cell maturation
Which three genes are used in a heavy chain locus
What about the light chain
Heavy:
V(variable)
D (diversity)
J (joining)
Light: only V and J
What is somatic recombination
Within each cell the V, D, J DNA sequences are randomly recombined during β cell maturation to generate a single V-D-J (or V-J) DNA sequence to form the variable arms of the antibody Y
What is an epitope
An antigenic determinant in a protein
What happens if a random beta-cell Happens to express an antibody that binds to an antigen of interest
The beta-cell will be stimulated to differentiate and multiply, producing a clone of beta cells, each secreting the same specific antibody against the antigen
Can more than one beta-cell be activated by an antigen
Yes
The blood from an immunised animal will generally contain a mixture of different antibodies to an antigen. Why is this and what is it called?
It is common for more than one beta-cell to be activated by an antigen
This mixture is known as a polyclonal antibody
How do you produce a mono monoclonal antibody
Beta cells must be removed from the spleen of an immunised animal and are fused with cells from a myeloma to produce hybrid cells that grow like cancer cells but produce large amounts of antibody
These antibodies are tested for antigen binding and the cell that produces the tightest binding antibody can then be selected and used to generate a cell line that manufactures large amounts of antibody.
Which are more expensive to produce:
Monoclonal antibodies or polyclonal antibodies
Monoclonal
True or false:
Monoclonal antibody is can be produced in unlimited quantities
True: parent hybridomas can grow indefinitely
What does ELISA stand for
Enzyme linked immunosorbent assay
How many wells are usually in the plastic plates used for ELISA tests
96 or 384
What do portable glucose testing kits use
How do they work
A disposable mini electrode that contains the fungal enzyme glucose oxidase and an electron carrier, ferrocene
When a sample is applied, any glucose present is oxidised by the enzyme but the enzyme is then diverted from its normal task of delivering the electrons to oxygen by ferrocene, which instead conduct the electrons to the mini electrode. This registers a current, proportional to the amount of glucose in a sample
How to work out P:O ratio in state 3
nmol of ADP added
——————————-
nmol of O consumed in state 3
What should be the P:O ratio be for
a) succinate
b) glutamate and malate
a) 1.5
b) 2.5
How can a theoretical P:O ratio be calculated
The number of protons injected during electron transport (H+:O)
—————————————-
The number of protons used to synthesise each ATP ( H+:P)
What is the number of protons pumped out of every oxygen atom reduced by a pair of electrons for NADH and succinate
NADH: 10
Succinate: 6
How do you compare the respiratory control ratio of a and B
Divide the gradient of a by the gradient of b
What are the values for the respiratory control ratios of the following:
A) glutamate + malate
B) succinate
C) ascorbate + TMPD
A) 3-8
B) 2-5
C) 1-2
Why might respiratory control ratios decrease overtime
The more mitochondria has been ill treated the more the membranes become leaky
In the mitochondria and metabolism practical, substrates with a higher proton:O ratio will have a ____ state 4 rate
Lower
What is DNP
An uncoupler - dinitrophenol
Describe an experiment to test the effects of inhibitors on mitochondria and metabolism
Wash vessel with water and ethanol Add medium and mitochondria and put lid on Add glutamate and malate Add excess of ADP Add rotenone Add succinate Add antimycin Add ascorbate and TMPD Add cyanide
Give some time between each addition to record the change in O2 usage
Why are glutamate and malate added first to the mitochondrial mixture when testing the effects of inhibitors
This produces NADH for oxidation by complex one
what does rotenone do
So what will happen to O2 consumption after its addition to mitochondria
What is added after this? Why?
Inhibits complex 1
It will dramatically decrease
Succinate as it is a substrate for complex 2
What will happen to O2 consumption when succinate is added to mitochondrial mixture after rotenone is added?
Drastically increases
What is antimycin
Inhibitor of complex three
How are ascorbate and TMPD related to oxidative phosphorylation
This forms a non-physiological substrate complex IV
What happens to oxygen consumption of mitochondria if cyanide is added
What would you expect to happen
Consumption decreases but does not stop
This is because non-enzymatic auto oxidation of ascorbate occurs
What does cyanide do
Inhibits complex four by poisoning cytochrome oxidase
Difference between serum and plasma
Plasma contains all the clotting factors
Both contain no cells or platelets
How do you get rid of cells from blood
Centrifuge
Why may absorption not literally relate to protein concentration when using Coomassie blue dye
What must be used because of this
What cannot be done
Why is any value only an estimate
The binding of the die is more complex than a 1:1 stoichiometry between dye and protein
A standard curve
You cannot extrapolate be on the highest value
This die binds to arginine and arginine content per molecule can vary
Some asses cannot differentiate between different proteins why would these be useful in clinical practice
If you are interested in work there is protein in urine as an indicator of kidney function
What is the unit of Katal?
What is it used to show
Moles per second
Efficiency of enzyme
Why would one bother to convert the activity data to katals per mg protein
To present in standard form so you. An compare efficiency of 2 different enzymes found from different assays
How to calculate Mr from SDS PAGE
Find Rf (migration distance of protein/ migration distance of dye)
Plot graph of Rf (x) vs Mr (y)
Estimate Mr from calibration curve
Why might SDS page not be able to inform you of the true Mr of a native protein
Proteins composed of different subunits of different sizes give to bands and you do not know which ratios of the two bands comprise the protein
Give three ways that non-denaturing PAGE reveals different properties of proteins compared to SDS page
1) Preserves multimeric structure and S – S bonds
2) activity of protein is retained and activity can be revealed if there is an appropriate way of converting product of enzyme activity into a coloured substance
3) I’m It does not necessarily reveal the Ama of the multimer since separation is based partly but not only on size
Why can you not observe NADH absorbance in gel
One cannot use UV light through a gel and cannot place a detector under each band
Which is more specific,: Bradford assay or Eliza
ELISA
How can you measure ATP production
Luciferin
In the presence of ATP, oxygen and luciferase, Luciferin undergoes a multistep oxidative decarboxylation to oxyluciferin to produce light
Use a standard curve of the light emitted
Why would ATP decrease if DNP is added
Not only is ATP not produced but the current ATP is hydrolysed
What does oligomycin do
Inhibits ATP synthase but also inhibits hydrolysis of ATP by DNP
How does calcium affect mitochondria
How can this be monitored
High external concentrations of calcium result in calcium uptake, stimulating formation of the mitochondrial permeability transition core which causes swelling as salt and water enters and eventually swelling ruptures the outer membrane and release cytochrome c
Decreased mitochondrial light scattering and thus decreased absorbance
What does cyclosporine do
What else has a similar effect
Blocks permeability transition pore (but Ca can overcome cyclosporine at high concentrations)
ATP also blocks pore
uncouplers
How do uncouples prevent calcium induced pore formation in mitochondria
They eliminate the PMF and therefore prevent calcium uptake into the mitochondria which is normally driven by the membrane potential
What will you see in the oxygen electrode when calcium is added to mitochondria
What happens if you add cytochrome c
First succinate must be added to stimulate respiration
There is an initial stimulation of respiration by calcium due to the increased membrane potential
Followed by an inhibition of respiration by high calcium due to loss of cytochrome c
Addition of XS cytochrome C then stimulates respiration because it is able to replace the lost cytochrome c
What happens if you add XS cytochrome c before the addition of calcium to the mitochondria
Nothing
The cytochrome C cannot cross the intact outer membrane
How does the addition of hexokinase affect the rate of respiration
Stimulates
It removes ATP and supplies ADP for ATP synthesis
The stimulation of ATP synthase allows protons back into the mitochondria and decreases the PMF, making the respiratory chain work faster
do oligomycin and DNP have the same effect on O2 consumption
Oligomycin inhibits the ATP synthase by blocking protons through the synthase. This means the PMF increases back to state 4 level which inhibits respiratory chain and reduces oxygen consumption
DNP equilibrates H+ across the membrane, reducing PMF to 0, stimulating O2 consumption
In a medium of mitochondria and glutamate and malate only why does ADP failed to stimulate respiration
What happens if you add arsenate after adding ADP?
There is no phosphate in the medium to act as a co-substrate for ATP synthesis
Respiration increases as arsenate can be a substitute for phosphate, allowing the formation of ADP – As (an analog of ATP)
Why is the rate of respiration after arsenate is added faster than if you added phosphate to a medium of mitochondria, glutamate + malate and ADP?
ADP-As Readily hydrolyses in water, recycling ADP and allowing apparently uncoupled respiration
You have a solution of mitochondria, glutamate+malate, ADP and arsenate.
ADP-As has been formed. What happens if you add phosphate?
Phosphate will outcompete arsenate
This does not happen immediately because the ADP has to be converted to ATP before state three will slow down state 4.
How does DNP affect O2 consumption when added to a mitochondrial solution with ADP and arsenate
Would act immediately To block ADP-As synthesis so O2 consumption would rapidly increase
Design an experiment to distinguish between a toxin that inhibits the respiratory chain, one that inhibits ATP formation or one that uncouples the mitochondria
1) Incubate mitochondria with glutamate and mallet then add ADP to start state 3
2) add mystery toxin
3) If it inhibits respiratory chain or ATP formation, oxygen consumption will be inhibited. An uncoupler will increase O2 consumption
4) if it is one of the former 2 responses, add an uncoupler (DNP)
5) If toxin is ATP formation inhibitor, uncoupling will stimulate rate again by removing PMF. If toxin is an respiratory chain inhibitor, uncouple it will have no effect on the rate as the respiratory chain is inhibited
How would you verify a mystery toxin is an uncoupler in mystery toxin experiment
Add the mystery toxin in state 4 and see an increase in rate
What is the respiratory control ratio (give the equation)
The rate of mitochondrial respiration in the presence of Pi and ADP (state 3)
—————————————————
Rate of respiration in the presence of Pi and ATP (state 4)
What is state 3
The rate of respiration of mitochondria in the presence of Pi and ADP
(When the system is at full pelt)
What is state 4
When all ADP is used up
What are the units you need to know
a= 10^-18 f= 10^-15 p=10^-12 n=10^-9 μ=10^-6 m=10^-3 k=10^3 M=10^6 G=10^9 T=10^12 P=10^15 E=10^18
How to work out how often an enzyme will be expected to arise in a genome
What to be careful of
How many base you pairs is it? 4^x
Be careful of their being an unspecified base pair (this can be anything) so it would be 4^(x-1)
Describe the method for alkaline lysis mini prep
Why is this done?
- Add SDS, RNase, and NaOH to bacteria
- Neutralise the substance
- Add buffer (K acetate/ acetic acid) - this causes cell debris and chromosomal DNA to precipitate while plasmid DNA reanneals and stays in solution
Why are SDS, RNase and NaOH added to bacteria during alkaline lysis prep
SDS denatures lipid membranes
NaOH denatures dsDNA
RNase digests RNA
What happens to the plasmid after it is purified by alkaline lysis
Digested with restriction enzymes and the DNA fragments are separated by electrophoresis
What is used to replicate DNA in PCR
DNA polymerase
Why is it better to use Taq in PCR than just adding new DNA polymerase after each heating
Adding DNA polymerase is expensive and tedious
How did the primers in PCR replication differ from primers in Actual DNA replication in the cell
PCR- DNA primers used
Cell- RNA primers used
U replaces which nuclear tide in RNA
Is U purine or pyramidine
T
Pyramidine
Why are DNA sequences sometimes added to the end
Which end
So the amplify target sequence can be cleaned to an appropriate factor
this sequence is cleaved by restriction endonucleases
5’
What does Chelex do
Binds metal ions that inhibit PCR
Describe the method for PCR
Place sample in a solution containing Chelex
Lyse cells by boiling and centrifuge to remove cell debris
Mix with Taq pol, oligonucleotide primers, the 4 deoxy riboNTPs and MgCl2 (the Taq co factor)
Begin temperature cyclinh
What is the best and quickest way to measure the size of a DNA molecule
How does this work
Gel electrophoresis
DNA is covered in negative charges so will migrate towards the positive electrode
Smaller fragments thread their weight through the sievelike gel matrix more rapidly than larger fragments
Describe the method of DNA gel electrophoresis
The DNA samples are loaded into Wells at one end of the gel
The gel is then run by applying an electrical field
The dye, ethidium bromide, is added and slots in between DNA base pairs
Why is GelRead better than ethidium bromide
Both fluoresce under UV but GelRead is: Less toxic
More sensitive
Which wavelength do all nuclear acid bases absorb light strongly at
260nm
What should the ratio of absorbance 260:280nm be
2
Why would the growth of E. coli without antibiotics be slow in the first 60 minutes
What happens after this
Why does growth eventually stop
This is the lag phase as the bacteria adapt themselves to the growth conditions
After this they enter the exponential phase during which they double in number approximately once every 30 minutes
They have reached a gross something factor such as depletion of an essential nutrient and/or the formation of an inhibitory product such as an organic acid
What is the effect of chloraphenicol on E. coli
It is a bacterial static antibiotic
It inhibits translation by preventing protein chain elongation by inhibiting peptidyl transferase activity of the bacterial ribosome
Hence absorbance of bacteria will not change as the bacteria as a present but just not growing
Which enzymes promote chloraphenicol resistance
How does this work
CAT (Chloraphenicol acetyltransferase)
Add an acetyl group to the antibiotic, preventing it from binding to the bacterial ribosome
Why does penicillin act different to chloraphenicol
It is a bacteriolytic agent
It is an irreversible inhibitor of transpeptidase preventing bacterial cell wall synthesis. It does lead to cell lysis.
Why might absorbance of bacteria decrease slower when chloramphenicol and ampicillin are added together
The bacteriolytic effects of ampicillin I slowed down by chloramphenicol restricting translation and therefore the ability for the cells to divide
Thus cell lysis takes longer
Why would a restriction enzyme with a recognition site of 5bp (GANTC) be useful when mapping plasmids of 3kbp
As N is unspecified, such sites will arise every 4^4 bp=256bp
Therefore this enzyme is not useful as it cuts too frequently
How useful is a restriction enzyme with the recognition sequence of eight base pairs in mapping a plasmid of 5kbp
4^8 = every 65.5kbp
This is too high so is unlikely to have any restriction sites
Why do bacteria restriction enzymes not cut their own DNA
Bacteria use methyltransferase to methylate A and C residues within recognition sites
What is the target of DNA methyl transferases
Cytosine
If you had a large plasmid or DNA fragment, what are the problems with this and what methods could you use to produce a restriction map(3)?
Suggestion by multiple restriction enzymes often generates too many bands
This can be circumvented by isolating the individual bands from a single digest and then digesting the bands with a second enzyme
Alternatively a southern blot of a multiple digest can be probed with individual radiolabel fragments from a single digest
Or
If you perform n cycles of PCR how many double-stranded molecules are made
How many strands are what you want
2^n
(2^n)-2n
What does dimorphic mean
Present in some people and not others
Why do we obtain a 150 bp fragment of DNA using primers that are designed to amplify the Alu insert when there is no insert present?
The Primers did not immediately flank the insert but I separated in the gene by 150 base pairs
Otherwise the product would be too small to distinguish
Suggest five reasons why a PCR experiment may fail
- The DNA quality and amount: you need DNA intact without any contaminants that might interfere with PCR
- Primer problems: Primers need to be at least 15 nucleotides along with a high enough percentage of GC content to increase melting temperature without creating problems of secondary structure
- Cycling parameters
- Buffer composition
- Type of thermostable polymerase
What are important qualities a primer the PCR should contain (5)
At least 15 nucleotides long
High percentage of GC content
No repeat motifs
Should not self associate
Both primers should have the same annealing temperature
What is important about the buffer composition during PCR
Needs to have access magnesium over dNTP as magnesium complexed with dNTPs is essential for dNTP incorporation
Mg2+ is also a cofactor first Taq and increases melting point of DNA
What are the results of the following in PCR
1) too little Mg2+
2) high Mg2+
3) too high Mg2+
1) no product
2) more and more non specific bands
3) no PCR product
Why might you get too many PCR products
How to fix it
Primers anneal to several sites on DNA so need to increase stringency
Increasing annealing temperature reduces primer binding to non-target sites
Optimise magnesium
What does the hardy Weinberg equation assume (5)
In a large population, mating is random, there are no mutations that affect allele frequencies, no migration between populations, no selection, and all genotypes produce with equal success
True or false
P 53 has a high concentration in the nucleus usually
False
Usually made on demand
What balances the translation and destruction of P 53
How does it work
What happens if the cell is stressed
A negative feedback loop governed by MDM2 (a product of p53)
MDM2 binds to p53 to allow proteolytic activity
P 53 can be phosphorylated so MDM2 cannot find and P 53 concentration rapidly increases
How is TP53 different to P53
TP53 is the gene
P53 is the product
Dark Giemsa bands represent more condensed AT rich regions whereas light bands mark less condensed GC rich region. Which region contains more genes (3)
Heterochromatin stain more darkly then regions of euchromatin
Large sections of constitutive heterochromatin play structural roles for example forming contromeres and these reasons sequences tent be richer in AT nuclear tides
Deamination create a mutual bias that turn methylated C into T so there is a pressure to maintain GC content of gene rich regions
Gene rich regions are regularly transcribed and therefore usually located in euchromatin
What do the colourings of Dark Giemsa bands mean?
Dark Giemsa bands represent more condensed AT rich regions whereas light bands mark less condensed GC rich region
Give the different classes it chromosome morphology
Telocentric: no p arm, only q arms
Acrocentric: very short p arm, normal q
Submetacentric: short p - half way between acro and meta
Metacentric: p and q almost equal
How to work out if a chromosome is gene rich or poor?
# of coding genes ————————— x 1 million # of base pairs
(X by 1 million to convert to Mb)
Then compare to rest of genome (5.7genes/Mb)
What kind of process could cause blocks of a chromosome to appear in a different order or even swap orientation
Double-stranded breaks, after which the fragments have been re-assembled in a different order
This could occur because of NHEJ
How would you know if a DNA break is paracentric
If the centromere isnt included
What does synteny do
Compares the genome of one species to that of another
Green Lines on the diagrams will link conserved genes
Black lines connect blocks that are the same orientation
Brown lines rink regions that possess opposite orientations
As far as synteny is concerned, which genome is most similar to human chromosome 17: chicken, chimpanzee, gorilla
All genetic material from chromosome 17 appears on a single chimpanzee chromosome, although much of it has been fragmented and reassembled
The gene order in human DNA is spread between two gorilla chromosomes but the blocks are not much altered
In the chicken the gene sequence is much more fragmented – it is shared between four chromosomes and has undergone multiple rearrangements
Gorilla>chimpanzee> chicken
What are paralogs
Genes that share high similarity
Eg a-Hb and f-Hb (fetal and adult Hb paralogs)
Give the key for the exon sequence you in bio Informatics
Empty box= 5’-3’ untranslated region
Filled box: exon
V shaped connecting line: intron
What is the difference between a missense valiant and synonymous variant
Synonymous mutation is change the code on sequence but do not alter the amino acid
Missense mutations code for different amino acids
What causes Li Fraumeni syndrome
What does this mean for inheritance
A single germline mutation in p53 So
It is autosomal dominant
CHEK2 on chromosome 22
If there is a mutation in both p53, does it make a difference from 1
Yes
Different rumours arise
What does CHEK2 also do
Phosphorylates P 53 to prevent MDM2 ubiquitin ligase binding, allowing [p53] to rise
How does LFS change between genders
Penetrance by age 50 is higher in females than males
Describe the DBD blue β sandwich
Four strands, all connected to the neighbour in an anti-parallel fashion
Describe the DBD red β sandwich
Seven strands, mostly antiparallel, but two strands (two and three) have a parallel connection
What feature Of the DNA target doesnt Arg-273 interact with
The positively charged side-chain of Arg makes favourable electrostatic contact with two negatively charged phosphate groups on the DNA backbone
What kind of interaction is Arg 248 Involved in and what feature of DNA does it target
What is this called
The side chain of arginine appears to hydrogen bond with a water molecule located in the minor groove of the DNA target
Intern the water molecule forms a hydrogen bond with a specific A base on the P 53 sequence
Water mediated minor groove interaction
Why does phosphorylation of Thr-18 weaken the interaction between P 53 and MDM2
Phosphorylation of the hydroxyl group in this Thr sidechain will introduce a larger group that this is more full negative charges. The interface between this portion of P 53 and MDM2 is mostly nonpolar so introducing such a large amount of negative charge is highly unfavourable
Explain why pharmaceutical companies might be interested in developing nutlins as anti-cancer drugs
Cancers that still possess unmutated P 53 deactivate it by up regulating MDM2 or knocking out the MDM2 inhibitor
Nutlin drugs act as re-activators of wild type p53
Why might completely knocking out MDM2 be negative
This may give gain-of-function P 53 mutants free reign resulting in tumour information
Stretches of GC rich self complimentarity are more problematic for PCR than AT which regions. Why
GC rich dimer regions have high melting points and are therefore had to disrupt which would exacerbate problems due to incorrect annealing in a PCR experiment
How do you do a southern blot
1) Take purified DNA and cleave it using DNase
2) gel electrophoresis
3) expose gel to a filter for a while and fragments will transfer
4) expose the filter to a radio labelled DNA strand that is complimentary to the desired strand
5) expose the filter to an x Ray
What are southern, western and northern blots used for
Southern: DNA
Northern: RNA
Western: Protein
What is the beer lambert law
Absorbance= molar absorption/ extinction coefficient x molar conc x optical path length
In what ways does non-denaturing PAGE technique reveal different properties of proteins compared to the SDS PAGE technique and why? (3)
Preserves the multimeric structure of proteins and S-S bonds
Because the protein is native it allows the activity of the protein to be retained and this activity to be revealed if there is an appropriate way of converting the product of enzyme activity into coloured substance
However it does not necessarily reveal Mr of the multimer since separation is based partly but not only on size
Describe method for ELISA
Coat plate with abundant amounts of antigen
Wash
Add new antibody at different concentrations
Incubate then wash
Add enzyme linked antibody that was raised against the original antibody
Add a substrate that will change colour when added to the enzyme
How does affinity chromatography work
Column material contains a molecule that specifically binds to the protein of interest
When the sample is passed through the column and washed only the protein of interest will bind - other proteins are washed away
Your acquired protein can be a looted from the column by using competitive ligand
Describe ion exchange chromatography
Use the column of charge material
Proteins bind to column with different degrees depending on their charge
Proteins are eluted with increasing salt which disrupts electrostatic interactions
Highly charged proteins will elute at high salt concentrations
What does gel filtration chromatography assess
Size - smaller take longer
Which method is used to asses how much protein
Which protein?
Quantifying levels of a specific protein?
Bradford assay
Gel electrophoresis (SDS PAGE)
ELISA
Give the 5 gels and their use in MIMS
Agarose: separates DNA fragments according to size
Formaldehyde agarose gel: RNA denaturing agent during agarose gel electrophoresis. Formaldehyde inhibits RNase to maintain RNA integrity
SDS PAGE: gel electrophoresis- protein separation by mass, uses polyacrylamide gel
Native PAGE: separates acidic, water soluble and membrane proteins in polyacrylamide gradient gel. No charge used so proteins are separated by charge
8M Urea polyacrylamide denaturing gel: denatures 2nd DNA and RNA structures and is used for separation by weight in a polyacrylamide gel
How to reach Katals from absorbance
Conc change per sec=
absorbance change per sec
—————————————-
Coefficient x path length
How to work out specific activity of enzyme
Katals
———————
Protein amount
What is PMF=?
ΔΨ - (2.303RT/F)Δph
How many ATP produced for each of the following
a) 1 molecule of glucose
b) glucose via anaerobic glycolysis
c) glucose via aerobic glycolysis
d) 1 acetyl CoA
a) 30 (5 from glycolysis, 25 from TCA cycle (2 pyruvate per 1 glucose)
b) 2 ATP
c) 5 ATP (2 ATP for breakdown and 1 NADH per Glyceraldehyde 3 phosphate - but energy is lost getting NADH out of Mt so the 2NADH only =1.5 ATP each)
d) 10 (3NADH+ 1 ATP + 1 FADH2)
Describe the method for Bradford assay
Pour acrylamide solution on separating gel. Allow to polymerise
Load samples and Mr markers into gel and apply current
How do you match acrylamide concentration to protein
Using a higher acrylamide concentration produces a gel with a smaller mesh size suitable for the separation of small proteins.
What may a band in PCR <100bp represent
Often v faint
Primer dimer
Primers that are not used up dimerise and ethidium bromide can intercalated - there is no PCR product corresponding to this band
