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________ ________ involves the stepwise removal of contaminants.
Can be analytic or preparative.
HOW DO YOU MEASURE PROTEIN PURIFICATION?
- Measured as an increase in specific activity of protein....ENZYMES...ACTIVITY.
- Ratio of amount of protein interested in over total protein)
PROTEINS ARE A RESULT OF WHAT 2 PROCESSES?
TRANSLATION AND TRANSCRIPTION.
WHAT HAPPENS TP PROTEINS AT HIGH/LOW IONIC STRENGTHS?
- LOW...proteins tend to remain in solution (salted in)
- HIGH.... protein solubility decreases...PRECIPATES OUT BY PELLETING IT OUT.
the most common salt used for precipitation of protein.
Ammonium sulfate is the most common salt used for precipitation of protein. WHY? NAME 2 !
Used because it is water soluble + high ionic strength salt
AS added gradually, different proteins of variable solubility come out of solution- dicard until your protein is precipitated
IN CHROMATIGRAPHY..The molecule’s rate of progress through the immobile phase is proportional to its affinity for the matrix
Components are fractionated between mobile and immobile phases...WHAT ARE THEY ?
- Proteins are dissolved in the solvent
- IMMOBILE PHASE IS WATER..HYDROPHILLIC. STAY BEHIND.
- MOBILE IS SALT...TRAVELS FATHER
WHAT THE HELL IS HPLC?
- High-performance (pressure) liquid chromatography
- Has a high resolutionLong narrow columns are used.
- Mobile phase is forced through a tightly packed matrix under high pressure.
- Fractions measured directly on instrument and can be collected for further analysis.
WHAT ARE A FEW CONDITIONS OF A PROTEIN THAT WOULD TELL YOU HOW FAST IT WOULD TRAVEL THRU THE MOBILE PHASE? NAME 4
- LIPIDS WITH IT
- REACTION TO SALTS
- Ip OF PROTEIN
- THESE ALL DEPEND ON THE PARTICULAR PROTEIN YOUR AFTER!
DEFINE Ion-exchange chromatography
- Proteins can be separated according to ionic charge.
- THINK Ip
- Proteins with charged groups associate with the column matrix
DEFINE Gel filtration chromatography
- Gel filtration separates proteins by molecular weight
- THINK SIZE EXCLUSION ..LARGEST 1ST)
- A gel filtration column is packed with cross-linked polysaccharides of different porosity Proteins that are small enough to enter the pores are eluted from the column last, each with specific hydrodynamic radius
WHAT CXHROMATOGRAPHY separates proteins by molecular weight?
DEFINE Affinity chromatography
Isolates one protein from a mixture. Specific ligand used (near totally pure).
IN Affinity Chromatographic procedure, THE PROTEIN YOUR AFTER...WILL IT COME OUT FIRST OR LAST?
- LAST. WASH OUT OTHER SHIT FIRST.
IN WHAT Chromatographic procedure DO YOU FIND DEAE CELLULOSE?
- Ion-exchange chromatography
DEFINE. TWO-HYBRID SYSTEM TO TEST FOR PROTEIN/PROTEIN INTERACTIONS.
DEFINE Polyacrylamide Gel Electophoresis
- Based on protein migration in an electric field.
- Composed of cross-linked acrylamide.
- Movement of proteins depends on molecular size, shape, and charge density.
- The position of proteins in the gel can be visualized by staining, autoradiography, or Western blot.
T OR F
polyacrylamide gel electrophoresis (PAGE), IT'S DONE BETWEEN TO PIECES OF GLASS.
THE MOVEMENT OF PROTEINS IN A POLYACRYLAMIDE GEL IS DEPENDANT UPON WHAT? NAME 3
molecular size, shape, and charge density
In SDS-PAGE, proteins are separated on the basis of their molecular weight only!!!
DEFINE SDS (FROM SDS PAGE)
sodium dodecylsulfate (SDS)
In isoelectric focusing, the gel consists of a mixture...NAME IT
- AmpholytesHave various ratios of positive and negative charges.
- Electrophoretic movement of ampholytes establishes a pH gradient.
- Proteins stop when they reach the pH equal to its isoelectric point
Two-dimensional gel electrophoresis Separates proteins based on WHAT?
- Isoelectric point and molecular weight
- Run gel in electric field in one direction via isoelectric point on a tube gel
- Then, place tube gel on SDS PAGE
HOW DO YOU RUN A TWO DIMENSIONAL GEL?
- Run gel in electric field in one direction via isoelectric point on a tube gel..TO GET Ip
- Then, place tube gel on SDS PAGE....TO GET molecular weight
NAME 3 TYPES OF GEL RUNS FOR PROTEINS.
- SDS PAGE...MW
HOW DOES MASS SPEC WORK?
- MEASURES MASS/CHARGE OF PROTEIN.
- POS BEAM OF IONS GOES THEU MAGNET. RECORDS THE DIFFERENCES.
You want to know if a culture of cells is in the process of DNA synthesis. You incubate your cells in the presence of radioactive thymidine to see if it is being incorporated into the DNA. What is the best technique to detect the labeled deoxynucleotide in nuclear DNA?
agarose gel electrophoresis
The major difference between defined and undefined cell media used in cell culture is.....
defined medium is free of serum, lymph or other fluids from living sources.
Which of the following would be the least promising source of cells for a primary cell culture?
A. embryonic tissue
B. adult rat brain tissue
C. plant protoplasts
D. actively growing malignant tumor tissue
B. adult rat brain tissue
(this multiple choice question has been scrambled)
Which type of column chromatography separates proteins on the basis of molecular weight?
A. ion-exchange chromatography
B. gel filtration chromatography
C. affinity chromatography
D. isoelectric focusing
gel filtration chromatography
In gel electrophoresis, the tracking dye moves:
A. more slowly than all the different molecules of the sample.
B. at the same rate as all the different molecules of the sample.
C. more quickly than all the different molecules of the sample. D. not at all.
more quickly than all the different molecules of the sample.
Nucleic acid hybridization can be used as a measure of evolutionary relationships between species.WHY?
Closely related species form hybrid DNAs with relatively high melting temperatures.
The role of the vector DNA in DNA cloning is to.....
identify the host cell that has taken up the one specific gene of interest
Why does one need to make replica plates when screening for a specific DNA sequence among a large number of recombinant bacterial colonies?
One wants a living culture of recombinant cells available after screening, a process that destroys the cells.
What is a “gene gun”?
gun that fires DNA-coated pellets into plant cells
Why are heat-stable DNA polymerases from thermophilic bacteria required for the polymerase chain reaction?
These enzymes are stable enough to withstand the temperatures required to melt DNA.