Flashcards in Lab Exam Deck (80):
What are fatty acids?
Comprised of two fatty acids and a phosphate group on a glycerol backbone
Hydrophilic head and hydrophobic tails
Essential for cell integrity and role in emulsification and absorption of fats
Crucial diagnostic and therapeutic value
Describe the amplex red assay
In presence of peroxydase, reacts with hydrogen peroxide in a 1:1 stociometric to yield highly fluorescent resorufin
Release of water molecule
Describe a carbohydrate
3,4,5, or 6-carbon atom chain where each carries a carboxyl group (except terminal)
Contain carbon, hydrogen, and oxygen which can reduce heavy metal salt solutions after hydrolysis
Exist in both cyclic and open forms
Cyclises by loss of water and formation of hemiacetal linkage between head grill and hydroxyl group on one of C atoms
Remaining groups can dehydrate to form glycosidic linkages
Describe the iodine test
Distinguish starch and glycogen
Iodine reagent forms dark blue complex with starch and brown-blue with glycogen
Iodine atoms fit into helacies to form complex
Starch is less branching therefore bind more iodine atoms = more blue
Describe the benedicts test
Distinguish between reducing and non reducing sugars
Red precipitate = reducing sugar (glucose, fructose, maltose, turanose)
No colour change = non reducing (sucrose, trehalose)
Reducing oxidized by Cu++ on solution to form carboxylic acid and reddish precipitate of copper (I) oxide
Describe the barfoeds test
Distinguishes between monosacchrides and disaccharides
Réduction of Cu2O
Rate at which blue precipitate appears is watched
2-3 minutes = monosacchride (fructose or glucose)
Disaccharide (maltose or turanose)
Describe the seliwanoffs test
Differentiates between ketohexoses and aldohexoses
When ketose is heated with a strong mineral acid (HCl) 5-hydroxynethymfulfural is formes
Forms red complex with resorcinol within 2 minutes (fructose)
Aldose gives colour more slowly (glucose)
Describe the anthrone assay
In solution aldoses equilibrate between open and closed
Treatment of oligosacchrides with concentrated sulphuric acid hydrolyses all inter ring links and oxidized each of the m monomers produced to a hydroxymethylfurfural
m oxidized monomers condense quantitatively with anthrone reagent to form m moles of coloured produce
What is beers law?
Concentration of original oligosacchride (c)
A = m(Ecl)
What occurs in the anthrone assay when treated with sodium borohydride?
Aldehyde group on terminal aldoses ring is reduced to a hydroxyl group
Only produces (m-1) oxidized monomers of coloured product
What are the two experimental difficulties of the anthrone assay?
1. If degree of polymerization or the identities of oligosacchride are unknown
2. If oligosacchride are longer than 6 units, difference between A and B become unreliable
What is recombinant DNA?
Using reverse transcription, a segment of mRNA is copied and amplified using PCR
Gene is inserted where restriction enzyme has cut
Undergo bacterial transformation into cell
Cell replicates and eventually replicated, producing multiple copies of DNA
What are the different types of vectors?
1. Mammalian expression
2. Bacterial expression
4. RNA interference
Describe mammalian vectors
Multiple cloning sites
Poly A tail
Advantages: post translational modification, can be used to localize fusion proteins, transfection reagents are expensive
Disadvantage: produce much smaller amounts of proteins than bacterial cells
Describe bacterial expression
Strong bacterial promoter
Multiple cloning sites
Ori of replication
Advantages: produce much larger amounts of protein then mammalian cells, easy to transform
Disadvantages: does not have post translational modification
What is an ideal plasmid?
Small, relaxed (present in multiple copies), compatible with host cell, and able to promote sexual conjugation
What are some important enzymes in recombinant DNA formation?
DNA ligases: catalyzes formation of phosphodiester bonds, DNA repair
- 3 types: E. coli DNA ligase (dépendant upon NAD+), bacteriophage T4 DNA ligase (dépendant upon ATP), mammalian ligase (dépendant upon ATP)
Restriction endonucleases: protect body from invasion by foreign DNA by degradation, recognize specific sequences by do not all cleave at specific DNA sites, useful in constructing recombinant DNA and other DNA derivative and in studying genetic structure and function
What are the 3 stages to a DNA ligase reaction?
1. Formation of a covalent enzyme-AMP intermediate linked to a lysine side chain in the enzyme
2. Transfer of the AMP nucleotide to the 5’ phosphate of the nicked DNA strand
3. Attack on AMP-DNA bond by the 3’-OH of the nicked DNA sealing phosphate backing and resealing AMP
Lac Z expression (b-galactoside)
Cloning of gene disrupts B-glactoside gene
Multiple cloning sites
Origin of replication
One HindIII site
Small size makes less susceptible to physical damage
High copy number and able to transform into recipient cells
Ampicillin and tetracycline resistant
No multiple cloning sites
Origin of replication
One hind III site
Small size makes them less susceptible to physical damage
High copy number and able to transform into recipient cell
How do bacteria protect themselves from their restriction endonucleases?
Bacteria mask their own DNA restriction sites by modifying them with methyl groups. These groups are added to adenine or cytosine bases, depending on bacteria type, in major groove. They do not block normal reading and replication. Each enzyme has a specific methyl transferase enzyme that protects the same sequence.
Describe the method for isolating plasmid DNA
1. Lysate preparation: form a pellet than resuspend using resuspension buffer, lyse cells using lysis solution, add neutralization solution to restore favourable conditions
2. Binding to column: h-bonds with column under high salt concentrations
3. Washing bound DNA
4. Elation bound DNA: binds with water when lower salt concentration
5. Assess purity using nanophotometer
What is the role of the resuspension buffers in the spin Column chromatography?
Tris HCl = buffer
EDTA = cheating agent, chelates Mg2+ which is required cofactor for many nucléases minimizing DNA breakdown
RNAase = breaks down DNA
What is the role of the lysis buffers in the spin Column chromatography?
SDS = detergent, dissolved lipids and degraded cell membrane
NaOH = denatures DNA including plasmid DNA
What is the role of the neutrilization buffers in the spin Column chromatography?
Potassium acetate: restores favourable pH conditions (lowering), allowing renaturing of DNA, precipitates SDS with lipids catching chromosomal DNA
Guanidine hydrochloride: denatures proteins and removes them with SDS bundle, leaving only plasmid DNA
Describe agarose gel electrophoresis
Repeating units of D-galsctose and 3,6-anhudrogalactose
Forms macroporous matrix which allows rapid diffusion of high molecular weight without significant resistance
Molecules travel towards negative anode, smaller ones getting caught in pores and slowing
What are the components of the loading dye for the agarose gel electrophoresis?
Sample and mix (buffer and HindIII) OR sample and h2O and loading buffer
What are the components of the loading buffer for agarose gel electrophoresis?
Glycerol: more dense then water and holds it down in the wells
EDTA: drops restriction enzymes from cutting
Why is gel red dye used in place of ethidium bromide in gel electrophoresis? How does it work?
Safe and more sensitive and stable
Does not penetrate cell
Fluoresence an orange colour when exposed to UV light that strongly intensifies after binding to DNA
Explain the action of ampicillin as an induced of plasmid replication
When the cells containing plasmids are cultured, they are grown on selective media with ampicillin to select transformants. Selective pressure allows bacteria to grow and maintain plasmid in the population by killing any cells not killing the ampicillin present cell in the plasmid. Allows for the isolation of plasmid containing cells and makes targeting more feasible
Describe nanodrop measurement
Can measure concentration and purity of proteins and nucleic acids with UV absorbance
Only requires 1-2uL of sample
1.8 = pure for DNA
2.0 = pure for RNA
Why is poly acrylamide gel electrophoresis not suitable for analysis of most plasmid DNA?
Polyacrylamide gel electrophoresis is not suitable for large plasmids. Can only read plasmids in native structure up to 2000bp and denatured structure up to 800bp, resulting in a limited view as plasmids can vary in size from 1Kb to 200 Kb in size. Only a very small portion of plasmids gal below 2000bp
What kind of chemical bonding holds cohesive ends together after action of restriction enzyme?
After the action of restriction enzymes, the base pairs of cohesive ends are held together through hydrogen bonding between base pairs on sticky ends of DNA
What are the basic steps involved in a recombinant DNA experiment?
1. Designation of target sequence
2. Amplification of DNA, replication and purification
3. Selection of corresponding restriction endonucleases
4. Encorperation of target DNA into vector DNA through ligase reaction
5. Transfer of recombinant DNA to host cell (électroportation/chemical competence)
6. Cells grow and duplicate, underground selective media (ampicillin)
7. Perform experiment (usually purity plasmid)
Describe the chemical basis of b-lactamase detection method use
Test of the presence of b lactamase in a change of colour. If b lactamase is present, it will hydrolyses starch-iodine-penicillin G solution added, producing penicilloic acids. These react with iodine, withdrawing the iodine from the starch-iodine-penicillin g solution, causing discolouration. This can indicate the presence of e.coli DH5alpha/plasmid as multiple copies produce a lot of b-lactamase, which renders its resistance to ampicillin
What is DHFR?
Ubiquitous enzyme involved in the biosynthesis of thymine nucleotides and hence DNA
Converts dihydrofolate to tetrafolate with the use of NADPH as the resultant
THF is a methyl group shuttle required in synthesis of purine, thumidylic acid, and amino acids
Describe the role of DHFR in cancer treatment
dTMP is a critical process for rapidly proliferating cancer cells
Interruption can kill those cells
Anti folâtra are potent inhibitors of DHFR by blocking regeneration of TMF and hence the synthesis of dTMP
What are the pros and cons of using eukaryotic cells in recombinant protein production?
Can glycosylate proteins
Post translational modifications
Proteins with large and complex structures
Isolation of proteins is usually easier
Growth medium is more expensive due to amino acids, vitamins, and growth factors required
Production runs are much slower as CHO cells divide more slowly
Greater chance of contamination
What are the pros and cons of prokaryotic cells in recombinant protein production?
Can produce large amounts
Low cost of growth medium
Sometimes do not fold properly
Cannot carry out post translational modification
Difficult to recover protein
What is the log phase of a cell culture?
Phase of exponential growth in cell colony
Describe the culturing of E. coli for protein expression
E.Coli contained pDHFR rehydrated and planted to generate individual colonies. A single colony is used to imitate growth of a larger culture grown to midlog phase. The stage expression of recombinant protein is induced by adding IPTG to the medium as the cells are dividing rapidly and protein production will be optimal.
What is the use of glucose in the medium while growing cells for recombinant protein production?
The glucose is added to ensure that the lac opéron remains repressed and no RNA polymerase or GST-DHFR-His is expressed
Leaky expression is undesirable as recombinant protein may be toxic and prevent bacterial growth
Describe the process for IMAC
Based on known affinity of transition metal ions to histidine and cysteine in aqueous solutions
Used for poly histidine tagged proteins
Eluated with imidiazole
Metal is bound to chelator incorporates into the matrix
Describe the équilibration buffer used in IMAC
5mM imidazole to prevent nonspecific bonding of any E. Coli proteins in solution that may contain histidine residues
Also contains Sodium phosphate and NaCl
Describe the wash buffer used in IMAC
10mM imidazole to remove any E. coli proteins that were able to bind, purifying the matrix so only the DHFR proteins remain bound
Also contains sodium phosphate and NaCl
Describe the elution buffer in IMAC
250mM imidazole to elute GST-DHFR-His protein by competent for the active sites, eluding the column
How was the DHFR activity measured?
Measure the rate of the reaction using a spectrophotometer that can detect light in the UV range
With every molecule of THF produced, one molecule of NADPH is converted to NADP+. NADPH absorbs light in the UV range at 340nm so when it is converted, the absorbance decreases as NADP+ no longer absorbs at the same wavelength
Define the specific activity
Amount of substrate the enzyme converts per mg protein in the enzyme preparation, per unit time
Define the total activity
Activity of an enzyme per milligrams of total protein
Describe the theory behind SDS PAGE
Size selective separation
Proteins move through gel in response to an electric field, allowing smaller proteins to travel more rapidly. Ina discontinuous gel, there are two sections, the proteins move more quickly through the resolving gel and then slow as they enter the stacking gel, forming a tight band which improves resolution. Proteins are separated in the presence of SDS and dénaturent agent, becoming fully dissociated and denatures from one another.
What is the purpose of SDS?
Binds non-covalently to proteins in a manner that imparts:
- an overall negative charge on the proteins, masking intrinsic charge of protein
- as a result, the rage at which the SDS bound protein migrates in the gel depends primarily on its size, enabling molecular weight estimating
Also gives SDS a long rod like shape in the proteins instead of a complex tertiary conformation
What is the matrix in the SDS-PAGE formed of?
Acrylamide monomers and bisacrylamide crosslinkers
4% stacking gel and 8-15% resolving gel
What are the components of the resolving gel?
Protogel buffer: stabilizes pH value to the desired value within the gel itself and in electrophoresis buffer
30% APS: source of free radicales and often used as initiator for gel formation
TEMED: stabilizes free radical and improves polymerization
What are the components of the stacking gel?
What is the purpose of glycerol and chlorine in SDS-PAGE?
Chloride ions in the gel run faster than the SDS-PAGE proteins to form an ion front. The glycinate ions form a front behind the proteins.
As the glycine moves from the the stacking gel at pH 6.8 to the resolving gel at pH 8.8, it’s charge goes from 0 to 1 and therefore travels more quickly. It compresses the proteins between the two ions, resulting in the thin band that is more focused.
What are some methods of quantification of proteins for SDS PAGE?
Simple optical density measurement
Describe the Bradford assay
Relies on coomassie brilliant blue G-250
Negatively charged due binds to positively changed amino acids (lys, his, arg). This shifts the absorbance from 465nm to 595nm. Detergents can result in high background interference.
Describe the lowery assay
Copper ions react with the peptide bonds under alkaline solutions. Oxidation of aromatic residues (tyr, trp). This requires the folin-crocalteu reagent which becomes reduced upon the oxidation of amino acids. Measured at 750nm.
Describe the biuret assay
Reagent is made up of sodium hydroxide, hydrated copper (II) sulphate, potassium sodium titrate. The copper ion forms violet-coloured coordination complexes with nitrogen in the peptide bind in an alkaline solution. The potassium sodium titrate is added to chelate and thus stabilizes the cupric ions. Has an absorbance measured at 540nm
Describe the BCA assay
Similar to the lowery assay. Peptide bonds reduce the copper ions from the curpic sulfate to Cu+. The amount of Cu2+ reduced is proportional to the amount of protein present in solution. Cys, tyr, trp amino acid side chains assist in the reduction of Cu2+ to Cu+. Two molecules of bicinchininic acid chelate with each Cu+ ion, forming a purple product that strongly absorbs light at wavelength of 562nm. It is less sensitive to detergent but more sensitive to reducing agents.
Describe the simple optical density measurement
Absorbance at 280nm (aromatic side chains trp, tyr) or 205nm (peptide bond) provides a rough approximation of protein concentration. Typically, 1A280 unit = 1mg/mL. If the protein sequence is known, the theoretical extinction can be estimated:
E280 = (#Trp)(5500)+(#Tyr)(1490)+(#Cys)(125)
Describe the principle behind the flurescence assay
Used to study protein folding/unfolding
When the protein unfolds, amino acids at the hydrophobjc center than fluorcence are exposed to the hydrophilic environnement and the fluorescence can be measured
What amino acids have fluorescent properties?
Phe, trp, tyr
Measured at 340/320nm
What is the use of SYPRO orange?
Has been shown to be useful in accessing protein thermostability in a systematic way. When heated, the protein denatures showing the core. The hydrophobic dye binds to the hydrophobic regions and fluorescents. The later aggregation of the proteins causes the dye to dissociate
Describe the theory behind western blotting
Seperation of protein mixture by gel electrophoresis
Provides molecular weight information on individual proteins
Can be stained on membrane for visualization
Proteins travel to membrane from negative to positive cathode
Proteins bind to membrane then primary antibody binds. Secondary antibody binds to primary and reporter (horse radish peroxidase) causes a reaction with peroxidase to produce a light.
Describe the sandwich of western blotting
Compare PVDF and Nitrocellulose membranes in western blotting
PVDF: good strength, 100-300ug/cm protein binding capacity, solvent resistant, uses coomassie blue, detection through chrimogenjc, chemoluminesent, fluorescent, chemifluorescence, and radioactive, can undergo double blotting, rapid immunodetection, reprobing, edman sequencing, and amino acid analysis, good binding in presence of SDS
N: poor strength, 80-100ug/cm protein binding capacity, no solvent resistant, uses SYPRO blot, detection through chrimogenjc, chemoluminesent, fluorescent, and radioactive, can undergo reprobing, and amino acid analysis good binding in presence of SDS
What are some factors affecting protein binding?
Found 80% decline in sequence efficiency of small peptides after hydrophobic residues were cleaved, presumably due to washout of the peptide remmetants
Peptides that characterize as hydrophobic often do not elute from as membrane as efficiently
Protein absorption results from the interaction of hydrophobic amino acid side chains and hydrophobic domains with the polymer surface
What are some factors effecting protein transfer?
Presence of SDS: useful in low amounts when being transferred have low stability in the absence of SDS, high MW may also exhibit solubility problems without SDS especially after denaturing conditions
Current and transfer time: critical for success, insufficient time/current results in insufficient transfer, if too high may migrate too fast to be absorbed
Transfer buffer pH: if isoélectric point = buffer pH, transfer will not be promoted
What are the functions of methanol in the transfer buffer?
2 major functions:
- stabilizes the dimensions of the gel
- strips complexed SDS from the protein molecules
Describe how methanol stabilizes the gel
Polyacrylamide is a hydrogel that has the capacity to absorb water, causing gel to swell. Methanol minimized swelling when addled to the transfer buffer. The lower the methanol the more time is required to reach equilibrium.
Describe how methanol strips SDS from protein molecules
SDS is necessary for resolution of individual proteins but can be extremely detrimental to effective blotting. The negative charge density imparted by SDS causes protein molecule to travel very rapidly through membrane, reducing resistance within pore structure and minimizing opportunity for molecular interaction. SDS also limits the ability of the protein to make molecular contact with PVDF
What is the purpose of the blocking step in western blotting?
The antibodies must only bind to the protein of interest and not to the membrane. An intert protein or non-ionic detergent can block unoccupied membrane sites, reducing non-specific binding (NSE). Blocking agents should have a higher affinity for the membrane sites than the antibodies so as to fill all unoccupied sites without displacing target proteins from the membrane.
What is immunodetection?
Uses a specific antibody to detect and localize a protein blotted onto a membrane permits the identification of a single protein in a complex sample. The proteins bind to the membrane, a primary antibody binds to the protein and the secondary binds to the primary. An enzyme on the secondary converts substrate to product, releasing light and allowing measurement.
What are the steps to immunodetection?
1. Blocking unoccupied sites to prevent NSB
2. Incubating membrane with primary antibody which binds to protein of interest
3. Wash to remove any unbound antibodies
4. Incubating membrane membrane with a conjugated secondary antibody
5. Washing to remove any inbound secondary antibody
6. Incubating membrane with substrate to reveal the location of the proteins
When can dry milk not be used in place of bovin serum albumin in the blocking step?
Cannot be used with biotinylated or concanavallin-labelled antibodies since it contains both biotin and glycoproteins therefore they cannot be used in avidin-biotin detection systems
What are some factors affecting immunodetection?
Washing: amount of washing
Double blotting: eliminates non specific binding
Detection substrates: chemogenic, chemiluminensence, chemifluorescent
Reprobing immobilon PVDF transfer membrane
How does the buffer effect immunodetection?
Must preserve the biological activity of antibodies. The ionic strength and pH should be relatively close to physiological conditions.
How do the antibodies affect immunodetection?
Advantages to secondary antibody: more than one able to bind multiple to single primary (amplification), can be used for a large number of primary, eliminating need to label numerous primary antibodies
Use polyconal or monoconal antibodies
Must not cross react with components of blocking buffer and be relatively pure
Concentration must be optimized