LAB TECHNIQUES Flashcards
1
Q
How are proteins and biomolecules isolated from body tissues/ cell cultures?
A
- cell lysis
- homogenization (crushing, grinding or blending of tissue)
- centrifugation (isolates proteins from smaller molecules)
2
Q
Electrophoresis
Protein Isolation
A
- use of a gel matrix to observe migration of proteins in response to an electric field
- anode side of gel has positive charge and cathode side of gel has negative charge
3
Q
What are characteristics of molecules that move quickly through electrophoresis gel?
A
- small
- highly charged
- placed in large electric field
4
Q
What are characteristics of molecules that move slowly through electrophoresis gel?
A
- large
- convoluted
- electrically neutral
- placed in small electric field
5
Q
Native PAGE Electrophoresis
Protein Isolation
A
- ‘non-denaturing’ gel electrophoresis
- used to analyze proteins in their native state
- useful for comparing molecular size or charge of proteins known to be similar in size
- complete protein can be recovered after analysis
- limited by varying mass to charge and mass to size ratios of cellular proteins
6
Q
SDS-PAGE Electrophoresis
Protein Isolation
A
- separates proteins based on molecular mass alone
- denatures the proteins and masks the native charge so that comparison of size is more accurate
- function protein can’t be recaptured from gel
- variable affecting protein velocity: E (electric field strength), f (frictional coefficient)
7
Q
Isoelectric Focusing Electrophoresis
Protein Isolation
A
- exploits the acidic and basic properties of AAs by separating on basis of pI
- gel has a pH gradient with acid gel at the positive anode, neutral in the middle, and basic gel at negative cathode
- electric field causes positive charged proteins to move toward cathode and negative proteins toward anode
- protein stops moving when pH = pI
8
Q
Chromatography
Protein Isolation
A
- tool that uses physical and chemical properties to separate and identify compounds from a complex mixture
- isolated proteins immediately available for identification/quantification
- more similar the compound is to its surroundings (polarity, charge, etc) the more it will stick and move slowing through surrounding
- preferred over electrophoresis when large amount of protein being separated
9
Q
What is retention time?
A
- variable used during chromatography
- amount of time a compound spends in the stationary phase – where the sample is placed to start process
10
Q
Components with high affinity for ____ phase barely migrate
A
stationary
11
Q
Components with high affinity for ____ phase migrate more quickly
A
mobile
12
Q
Column Chromatography
Protein Isolation
A
- column filled with beads as an absorbent and gravity moves solvent and compounds down the column
- size and polarity play a role in how fast compound moves down column
- the less polar the compound the faster it can elute through the column (short retention time)
- can also be used to separate nucleic acids
- more similar the compound is to the solvent (mobile phase) the more quickly it will elute
13
Q
Ion-Exchange Chromatography
Protein Isolation
A
- beads in column are coated with charged substances so beads attract/bind compounds with opposite charge
- after compounds have moved through the column, must use salt gradient to elute charged molecules stuck in column
14
Q
Size-Exclusion Chromatography
Protein Isolation
A
- beads in column contain tiny pores of various sizes which allow small compounds to enter the beads and slow them down
- large compounds can’t fit in pores so travel through column quickly
15
Q
Affinity Chromatography
Protein Isolation
A
- coat beads with receptor or ligand that targets protein of interest
- can elute protein by washing column with free ligand or a receptor for the protein of interest, or can elute with specific pH or salinity level that disrupts protein/ligand bonds
- drawback of elution step: recovered substance can be bound to eluent
16
Q
What is used to determine protein structure?
A
- xray crystallography
- NMR
17
Q
What is used to sequence amino acids?
A
Edman degradation (uses cleavage to sequence proteins of up to 50-70 AAs)
18
Q
Bradford Protein Assay
Protein Isolation
A
- used to determine protein concentration
- uses a color change from brown-green (acidic form) to blue (basic form)
- increased protein concentration corresponds to larger concentration of blue die in solution
- most accurate when only one type of protein is in solution
- limited by presence of detergent in sample or excessive buffer
19
Q
List the 3 steps involved in Gene Cloning
DNA Biotech
A
- choose a plasmid (extra-genomic, small, circular pieces of DNA)
- treat the plasmid and the gene to be inserted with a restriction enzyme
- the “sticky” ends of the plasmid and gene stick together – the attachment forms a complete circle of recombinant DNA that the host bacteria will use to create new gene products
20
Q
PCR
DNA Biotech
A
- way to amplify a small piece of DNA exponentially
- after n cycles, there will be 2^n daughter strands
- uses thermostable polymerase (like Taq) because it can withstand the high temperatures used during this process
21
Q
What are the 3 steps used in PCR?
A
- Denaturation – original parent strands of DNA are separated at 94-96°C, H bonds between parent strands break
- Annealing (Primer Hybridization) – primer selects specific part of DNA to be replicated and binds to it, ideal conditions at 50-65°C
- Replication (Elongation) – thermostable polymerase creates new DNA, ideal conditions at 70-80°C
- REPEAT
22
Q
What things are required for a PCR reaction to occur?
A
- primers that are complementary to DNA in region of interest
- nucleotides (dATP, dGTP, dCTP, dTTP)
- DNA Polymerase from bacteria
23
Q
Gel Electrophoresis
DNA Biotech
A
- allows for determination of the length of a segment of DNA (in base pairs)
- can determine length of sample DNA by running a standardized DNA ladder in one well which has multiple migration points in the gel that signify specific lengths in base pairs
- gel acts as mesh barrier so smaller pieces of DNA can mover faster than large pieces
- charged electrode is on side of wells and positive charge is on far end of gel
- DNA is negatively charged so it moves toward the positively charged anode side of gel that is created by a current running through the gel
- can also be used to see if recombinant DNA as been inserted into the plasma correctly
24
Q
Southern Blotting
DNA Biotech
A
- used to detect presence and quantity of various DNA strands
- separates DNA fragments by length and then probes for a segment of interest
- DNA is blotted with a thin sheet of nitrocellulose “paper”
25
What are the 4 steps of Southern Blotting?
1. bathe sample DNA in restriction enzymes to break strand into segments [Transfer Buffer]
2. Gel electrophoresis is used to separate segments by size [Agarose Gel]
3. sheet of nitrocellulose paper is used to blot the DNA segment out of the gel and onto the membrane [Capillary Blotting]
4. hybridization probe placed on membrane to determine if the desired piece of DNA is present in the experimental sample (if use fluorescently labeled DNA for hybridization then nucleotides in the probe are tagged with fluorescent phosphorous so probe and other DNA segments that may have attached are easy to locate)
26
Northern Blotting
- method of detecting specific sequences of RNA by hybridization with cDNA
- use of gel electrophoresis, transfer to a membrane, and detection with a hybridization probe
27
Western Blotting
- used to identify specific amino acid sequences in proteins
- proteins are separated by gel electrophoresis on an SDS-PAGE gel and detected using labeled antibodies specific to the protein or part of the protein
- use of SDS-PAGE gel, transfer to a membrane, and detection with an antibody probe
28
What mnemonic can be used to remember blotting techniques?
```
*SNOW DROP*
Southern DNA
Northern RNA
O O
Western Proteins
```
