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105 Cards in this Set
- Front
- Back
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Before a new research experiment a researcher must ________
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perform a comprehensive literature review of the topic
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The first step to a project is often ______ but the specifics can change from project to project
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analyzing the DNA sequence of interest
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_______ can be used to analyze the DNA
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Bioinformatics
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The simplest way to synthesize a protein from amino acids is by using ________
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organic chemistry
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Organic chemistry can only synthesize proteins that are ____ in length but are useful for obtaining ____ peptides like a single alpha-helix
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~50-70 aa
short |
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Molecular biology uses ___, ___, ____ to express proteins
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PCR, cloning, bacteria
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Recombinant DNA
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DNA from various species that is not naturally found in the environment
(plasmid produced for project) |
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It is important that proteins are only created when desired so that there is a high enough _____ to produce a large amount of protein
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population density
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The expression of recombinant protein may ____ or ___ the bacteria
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halt growth
kill |
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Several protein expression systems utilize the ___ operon
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Lac
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In the absence of lactose the Lac operon is ____ and the _____ binds to the operator, preventing ______
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off
repressor RNA polymerase from transcribing the mRNA required to break down lactose |
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The Ptac expression vector has a _____ promotor that is silenced by the ______
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Ptac
lac repressor protein |
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The system is turned on by the addition of ____
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IPTG: a lactose variant that cannot be metabolized
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Upon IPTG addition the _____ is removed from the operator and RNA polymerase can ______ and E. Coli ribosomes can synthesize the protein of interest
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lac repressor
transcribe the gene of interest |
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The Ptac expression vector can be "______" meaning that _____
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"leaky"
it is more likely to have low levels of protein expression, even in the "off" state |
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This leaky-ness can result in ____ protein since many proteins are expressed at lower temperatures, or the protein may ___ the bacteria if it is toxic to them.
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misfolded
kill |
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The page expression vector has _____ control
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tighter
and therefore not as much background expression |
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Specific strains of E. Coli, like ____, carry bacteriophage ____ within their genome
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BL21
T7 RNA polymerase |
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Upstream of this is the ____ which is bound by the repressor , preventing the use of the promoter and preventing protein _____
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lac operon
expression |
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____ is used to turn the system on and remove the repressor and E. Coli ribosomes are used to make _____
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IPTG
T7 RNA polymerase |
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T7 RNA polymerase can then target the _____ on the plasmid and transcribe _____
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promotor
the gene of interest |
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The end result of the phage expression vector is ____
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high expression of a recombinant protein
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The ____ (cell free) system involves only a ribosome and the mRNA of interest
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in vitro
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The invitro system is very ____ but is expensive and produces a ___ amount of protein
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fast
low (mg) |
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Most labs that express proteins use _____
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prokaryotic cells
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Prok. cells take up the DNA ____ and divided ____
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easily
quickly |
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Bacteria are very easy to grow in culture and ___ can be produced in a day at low cost
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liters
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Prok, cells bring about ____ amounts of protein expression
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high
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They also allow for the expression of proteins that may be ____ to eukaryotic cells
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toxic
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The clone carrying the gene can also be selected for by using ____ and ______
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antibiotics and blue/white screening
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Sometimes in bacteria systems a protein may not be _____ correctly and have ______
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folded
inclusion bodies (misfolded protein aggregates that are not biologically active) |
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____ cells and ____ cells can produce larger amounts of proteins that may be more likely to fold properly and contain proper modifications
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yeast and plant
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These systems can be more difficult to work with than bacteria as the ________ involved can be more tedious and time consuming and the cells divide more ___
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DNA manipulation
slowly |
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One of the most efficient animal cell systems uses _____ to infect ____ with the gene of interest
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baclovirus
insect cells |
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This system allows for the expression of ____ proteins, high ____, proper ____ and post-translational modification, disulfide bond formation, oligomerization, and simultaneous expression of multiple genes
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very large (up to 5000 aa)
yield folding |
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The disadvantage to the baculovirus system is that ____
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it is not as straight forward as those in bacteria and it is slow (2 weeks) and expensive
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Mammalian cells can be used as a starting material for ______ by obtaining large amounts of animal organs
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protein purification
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This works well with ____ but is not feasible to obtain a large amount of ____ protein
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animals
human |
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This technique results in a final protein that is in its _____ state in that it is folded properly and contains the exact post-translational modifications
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native
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Mammalian cells are ___ to work with and produce a ___ yield of expressed protein
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hard
low |
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The fastest methods to protein expression: ____ & ____
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Yeast and bacteria
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Best method for proper folding : ___ & ___
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mammalian and transgenics
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Cells can be ____ using many techniques
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lysed
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______ can be used for breaking up euk. cells or organs
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mortar and pestel
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You can ___/___ for 3 cycles since the ice crystals disrupt cell membranes
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freeze/thaw
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Chemicals such as ___ or _____ can be used to disrupt the bacterial cell wall or cause _____
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detergents or organic solvents
osmotic shock |
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Sound waves can be used to break up cell walls and cell membranes in a process called ____
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sonication
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A ____ can be used to place cells under high pressure (40,000 psi) to destroy the cell wall cause internal proteins and DNA to be released into the buffer
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French press
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____ and _____ are the most used to break up and remove DNA and other impurities prior to loading the sample into a column
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sonication and centrifugation
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Once lysed, proteins must always be kept on ___ and purified immediately to avoid ___________
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ice at 4 degrees C
protease degradation, oxidation, and aggregation |
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_____ should be avoided after lysis as ice crystals can destroy proteins
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multiple cycles of freezing/thawing
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Preparative Purification
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Mass production (insulin, enzymes)
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Analytical purification
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identification, quantification, post-translational modification, protein interactions, structural studies
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Earlier technique of protein purification prior to chromatography
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Ammonium Sulfate cut
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Ammonium sulfate cut purifies proteins from ____
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organs or cells
sometimes chicken gizzards |
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In Ammonium sulfate cut, a _____ is used for controlled precipitation
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gradient
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Fractions of ________ are used
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10% 20% 30% 40% 50%
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A certain amount of salt is needed but if you have too much than ______ occurs and you will precipitate out your protein
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salting out
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Advantages of Ammonium sulfate cut
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isolate new proteins, no costly or difficult cloning/PCR, and protein is from its natural source (modifications)
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Current methodology for protein purification
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recombinant DNA technology
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Advantages of Recombinant DNA technology
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can obtain protein in large amounts, you manipulate the gene sequence, cost-effective,
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Disadvantages of Recombinant DNA technology
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must know DNA sequence, cloning/purification can be tricky, proper folding, no post-translational modification
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Advantages of In vitro
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No transformation, cell culture, expression, proteases.
High purity, no column required. fast (4 hours), newest kits claim to produce enough protein for sturctural studies (5mg) |
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Disadvantages of In vitro
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must have cDNA or mRNA; current production levels are on the order on micrograms; limited protein size (250 kDa); kit is expensive ($500)
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The _____ chromatography machine is more efficient than BioRad because it uses pistons for pressure control
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AKTA
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The more you purify a sample, the less ____ you get
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protein
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Have to make choice between : _____ and _____
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amount and purity
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In a protein dialysis you remove ___ from the sample
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salt
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You need ___ concentrations to see proteins on a gel
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high
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Disadvantage to centrifuge is that you cannot _____
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watch the sample in real time
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To store proteins you can put it in the refrigerate (4C) for ____
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a month
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Or store it at ___% or in ethylenel/glycerol at ___C for a year
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25-50%
-20 (freezer) |
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Best method of preservation :
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dialyze into buffer and dithiothreitol (DTT), store 100 microliters aliquots in freezer so only small fractions are removed from the cold at a time
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It is smart to add ____ to protect your sample from being infected with bacteria and fungus
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antimicrobial agents
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DTT =
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betamercapto ethanol
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____ involves loading a protein sample into a column which contains beads that bind to or separate proteins and collecting the fractions that drip out of the bottom of the column
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column chromatography
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Proteins can then be identified using a _____ which records the absorbance of the solution coming off the column at 280 nm
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chromatogram
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Proteins can be ____ using a SDS-PAGE
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identified
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SDS-PAGE involves ____ the protein and running it through a porous ___ to separate it by ___
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denaturing
gel size |
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____ is a process in which proteins are placed in a special membraneous tubing with very small holes that only allow the buffer through
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dialysis
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Dialysis is aka ____
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buffer exchange
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In dialysis ___ can be removed from a sample and ___ can be transferred into the buffer of choice
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salts and other unwanted molecules
proteins |
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Dialysis is usually performed at ___C and the buffer is often replaced at least once to ensure complete ___ of the buffer
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4
equilbration |
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Dialysis can also be used to purify ___ proteins from ___ proteins
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large
small |
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To distinguish: choose a tubing with a ____ cutoff between the larger and smaller protein
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molecular weight
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This allows the ___ proteins to pass through the membrane
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smaller
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To determine if two proteins interact using dialysis: ___
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if two small proteins bind one another then their combined mass will make them too large to pass through the pores of the tubing
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Protein concentration involves removing some of the ___ while keeping the ___ of interest
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buffer
protein |
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Samples may turn ___ as protein concentrations increase
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yellow
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Precipitation of preens out of solution will occur, to counter or reverse this increase the ___ concentration
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salt
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Proteins can be concentrates using lyophilization where they are ______
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dried under a vacuum over several days to a fluffy powder that can be stored in the freezer for years
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Ultrafiltration involves placing the proteins onto a ___ that allows only buffer to pass through
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membrane
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You can also concentrate proteins by running them through a ___ to which they bind and then ___ them in the volume desired
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column
eluting |
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REverse dialysis can also be used to concentrate proteins by placing the sample in dialysis tubing and and surrounding it in ____ to draw out the ___ and reduce the ___ volume
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polyethylene glycol
water buffer |
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Proteins may be frozen a ___ time
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single
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A protein sample should be kept in many small tubes (100 micro liters) so that ____
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you are not thawing out the entire stock when you need a small sample
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Homology alignment
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tells you what is important about aa and their domains
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XE projon
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machine that expresses a protein; takes the template dna inot a machine to purify protien, 90% pure proteins in 6 hrs
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BLAST research
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taking protein sequence entering it into a computer box and searches for similar proteins
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Have to induce bacteria at OD606 of 0.02 to 0.06 range because
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if not in this range then it is not optimal and will not express proteins
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E. coli cell lines used to take up ___ DNA
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foreign
no proteases, grow easily, easy to regulate |
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T7 lysozyme will degrade ___ and prevent ____
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T7 RNA polymerase
background expression |
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HSP90 Purification isolate it from ___
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nuclear extracts of halacells through ammonium sulfate cut and IEX
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Ultrafiltration can be done using ____ tubes ir ___ pressure in a stirred cell
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centrifuge
nitrogen |
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Insulin purification uses ___ chrom. and a ___ table
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metal ion affinity
purification |
