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60 Cards in this Set
- Front
- Back
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What is the molecular biological definition?
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mechanism, information, gene
To understand these mechanism of information exchange within the cell. |
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What is PPE for?
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Personal protective equipment used as a barrier method
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What is MSDS?
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Material Safety Data Sheet on chemicals
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What is OSHA and their requirement?
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Occupational safety and health Administration
Requires: written hazard communication MSDS, Labeling hazards property and handling and the proper training |
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What are the 3 types of pipette?
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TC- exact amount (to contain)
TD- To deliver by gravity TD/blowout- to deliver & fluid remaining @ tip is blown out |
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1000micro/liter =
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1mL
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Centrifugation
Analytical & preparative |
Analytical- use only small amount of material & studies the sedimentation characteristic of pure macromolecules
Preparative- most common used, separate large amount of materials |
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High speed vs. Large capacity
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High speed: used to collect microorganism, cellular debris
25,000rpm Large capacity: used for substance that sediment rapidly 6,000rpm |
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Rotors:
Fixed angle & swinging bucket |
Fixed angle: when centrifuged, particle separates @ an angle
Swinging bucket:when rotating, the tube is perpendicular |
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What does Spectroscopy mean?
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Light that are put into its component wavelength
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Fluorescense
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use to study biological system
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Infrared spectroscopy (IR)
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Less precise, can predict the substituent group (qualitative analysis)
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NMR (nuclear magnetic resonance)
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Used in organic to measure the spin state of proton by magnetic field.
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Spectrophotometer
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measure the intensity of a specific wavelength as it pass through a sample and compares it to a blank sample.
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Absorbance Vs. Transmission Vs. Concentration
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A= the amount absorbed by the sample
A=abc or A=log (1/T) b=pathway and c= concentration T= amount that pass through the sample T=Io/I Io=initial light I=final light Concentration: is proportional to absorbance and as T goes Up concentration goes down |
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260nm = ?
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DNA absorbance
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280nm =?
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Protein absorbance
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Why are various wavelength used?
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b/c difference samples absorbs at difference wavelength
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what allows us to see protein ?
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Ultraviolet light
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Amino Acid analysis allows us to do what?
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to determined the concentration of a pure protein
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Colormetric methods measure what?
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it measures the changes in color . As the protein concentration increases so does the intensity of the color.
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Bradford is used what?
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Used coomasie brilliant blue dye
Red=265nm Blue=595nm it is nonlinear and runs w/ a standard curve Used as a flow through check |
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280nm is what for protein?
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it's protein maximum absorbance
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Lowry is what?
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it is a common technique that measure the pure protein concentration
It is linear when we have low concentration of protein and used Cu+ |
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Dilution :
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M1V1=M2V2
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Variables does what?
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it measures and controlled in the experiment.
There are: Independent: variable you manipulate Dependent: changes when independence changes Control: kept constant |
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Enzyme overview:
What's the enzyme function? |
it is a catalyst that lowers the activation energy
it is highly specific are protein & RNA enzyme |
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Activation energy is what?
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it is energy need to overcome the reaction in order for it to proceed.
It transform to transition state It works by stabilizing the TS of chemical rxn and decreasing Gibbs free energy |
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What is the standard curve used for ?
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it graphs the relationship between absorbance and concentration
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what is the application of standard curve?
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Do not use values that are not in sync.
Avg. Abs. on plot on Y axis Conc. is plot on X axis create best fit line The result is the TOTAL protein concentration. |
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Enzyme Assays
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measure the conversion of substrate to product
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protein production can be done in what types?
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in vitro (free cell)
prokaryotic cells, yeast cells, plant, insect, mammalian cells. |
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what are the best protein production types?
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bacteria and yeast
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What are the three techniques for protein production/purification?
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1. Early technique- purify protein from cells or organs (blender)-little protein
2. Modern technique- recombinant DNA technology: pcr, cloning,-large amounts of protein 3.Newest technique- Cell free mRNA to protein in a tube q/ ribosomes. get high purity but costly |
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Protein purification:
Preparative vs. analytical |
Preparative: to mass produce
analytical: used to identify, quantify and study its structure |
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Ammonium Sulfate Cut
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helped controlled precipitation, where it clump. This allows us to check for the protein
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Dialysis
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putting the protein into a porous bag and then placing the bag in a solvent so protein can separate and migrate through the pores to reach equalibrium
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Protein concentration
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where nitrogen pressure is applied to the column and protein is being pushed out of the membrane and is watered out
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protein sequencing
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uses the N-terminal using Edman Degradation
sample is pure and analyzed each fragment with mass spect. to rebuild the protein |
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Calorimetry
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measurement of heat change when two protein interact.
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Biacore
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Immobilizing protein on membrane and run peptide across it.
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Size Exclusion chromatography
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separation of analyte
Immobized phase is sationary phase Mobile phase is Effluent |
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How does separation occurs in SEC?***
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separation is based on size. Large is eluted 1st then med. then small. ***
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What is the relationship between the SEC and the UV chromatogram and SDS-PAGE?
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Since the largest protein elute first we should see the absorbance increase when protein concentration increase on the graph. As for SDS-PAGE, it is reverse, small protein elute first and large protein remains in the stacking gel.
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Ion Exchange Chromatography
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use to separate proteins based on molecular charge
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in IEX protein in the solution that are supposed to be absorbed have what?
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net charges that are balanced by counterions.
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What is the relationship between pH, pI, and protein charge?
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pI is equal to pH, where all charges are equal.
If pH > pI = (-) charge If pH < pI = (+) charge So if Ph is greater than pI it is negative, if pH is less than pI it is positive charge for the protein. |
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Anion Exchanger
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Positive charge beads
Positive protein elute first Counterion is Chlorine |
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Cation Exchanger
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Negative charged beads
Negative protein elute first Counterion is Sodium |
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Affinity Chromatography (AC)
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used to purify specific biological macromolecules such as protein and nucleic acids
highest form of chromatograph . use antibodies bound to an inert material. |
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Hydrophobic interaction Chromatography (HIC)
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uses the differences in hydrophobicity on the surfaces of the protein to separate the proteins, where sample can be applied to a column that is highly polar= increase interaction of hydrophobics. sample can then be eluted out with solvent that is either polar or nonpolar.
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Gene fusion technique:
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MBP is attach to protein of interest than runs through amylose column and washed. X-factor will cleave protein
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Metal-Chelate chromatography
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gene of interest is encoded w/ histidines tag, & his-tag like nickel, we uses immobilized nickel to bind to the protein. Imidazole is used to elute the fusion protein b/c it competes w/ his-tag.
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What is the SDS-PAGE concept?
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SDS will cause the protein to have an identical charge-to-mass ratio. making all protein Identical (-). & therefore is only based on size
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what does the B-mercaptoethanol do?
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It denatures the protein and let the SDS binds to protein to make it more negative.
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Acrylamide pores can be controlled with what?
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The amount of acrylamide used and the degree of cross-linking
High degree of crosslinking = narrower pores mainly control the acrylamide |
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Gel electophoresis direction of movement is ?
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from positve(anode) to negative(cathode). Large & highly positive more slower than Large& low positive.
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What is the point for the stacking gel?
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Causes the protein to be concentrated into a narrow zone at the top of the resolving gel.
stacking gel contains less acrylamide and resolving gel contains more acrylamide |
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What is 2-D gel?
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It is a combination of isoelectric focusing and SDS-PAGE, where it is first separated based on its pI then by its molecular mass is SDS-PAGE
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What are the steps for PCR?
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1.template denaturation
2.Primer annealing 3.Primer extension (elongation) |
