Cell Biology Lab Final

Front 1

Requirements for standard curve:

Back 1

-Pure sample of known concentration
STANDARD
Bovine Serum Albumen (BSA)

-Method to assess the samples being measured
READOUT
Spectrophotometer (UV, visible)

Front 2

The components of an assay are:

Back 2

Accuracy
Precision
Sensitivity
Specificity

Front 3

Coomassie has a ____ charge allowing it to bind to ____ charged amino acids which causes ________.

Back 3

negative
positive
change to a blue color, absorbing light at 595nm

Front 4

_____ extraction will precipitate protein and nucleic acids will partition into the
aqueous phase.

Back 4

phenol/chloroform

Front 5

This strips protein from nucleic acids

Back 5

phenol/chloroform

Front 6

_____ will come out of solution (“precipitate”) in the presence of salt and ethanol

Back 6

nucleic acids

Front 7

Allows “purification” of the nucleic acids so that UV absorption will not overestimate the DNA/RNA

Back 7

salt and ethanol

Front 8

Nucleic acids will come out of solution (“precipitate”) in the presence of:

Back 8

salt and ethanol

Front 9

Ribulose 1,5-bisphosphate carboxylase (RUBISCO) does this:

Back 9

catalyzes the fixation of CO2 on carbohydrates in the calvin cycle

Front 10

Ribulose 1,5-bisphosphate carboxylase (RUBISCO) fixes CO2 to carbohydrates at a rate of:

Back 10

3CO2 /sec

Front 11

Ribulose 1,5-bisphosphate carboxylase (RUBISCO) has ___ subunits made up of:

Back 11

16 (550kDa)
8 large (52kDa)
8 small (13kDa)

Front 12

Cell fractionation was accomplished by:

Back 12

Homogenization and Centrifugation

Front 13

With differential centrifugation, these speeds are used to obtain the corresponding organelles

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• 600 x g
– nucleus
• 10,000 x g
– Mitochondria, lysosomes
• 105,000 x g
– Golgi and ER membranes

Front 14

The procedure for differential homogenization in experiment 3:

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• Homogenize
– Crude Homogenate (CH)
• Centrifuge #1
– 200 x g
-Low Speed Supernatant (LSS)
• Centrifuge #2
– 1000 x g
-High Speed Supernatant (HSS)
-High Speed Pellet (HSP)

Front 15

THINGS NEEDED FOR A CLONING EXPERIMENT

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•DNA fragments to be cloned
•A cloning vector (PLASMID)
•Restriction Enzymes
•DNA Ligase

Front 16

PLASMIDS
•ESSENTIAL FEATURES

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–Origin of replication
–Antibiotic resistance gene

•ADDITIONAL STUFF
–Bacterial or Eucaryotic promoter
–Eucaryotic drug resistance
–Other modification signals

Front 17

HOW DO YOU CUT THE PLASMID TO INSERT A PIECE OF DNA INTO IT?

Back 17

Restriction enzymes

Front 18

Bacteria express specific enzymes that physically cut DNA at specific sequences at this length:

The enzymes are called:

Back 18

4-8bp
restriction endonucleases

Front 19

RESTRICTION NUCLEASES
(aka: Restriction Enzymes)

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Naturally occurring BACTERIAL enzymes that cut DNA at the phosphodiester bond only at DEFINED SEQUENCES.

Front 20

Naturally occurring BACTERIAL enzymes that cut DNA at the phosphodiester bond only at DEFINED SEQUENCES.

Back 20

RESTRICTION NUCLEASES
(aka: Restriction Enzymes)

Front 21

HOW DO YOU JOIN DNA?

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*Cut DNA with RESTRICTION ENZYMES
*Add fragments cut with same enzymes
Add DNA LIGASE
*DNA will be covalently connected

Front 22

APPLICATIONS of restriction enzymes:

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*Cut the large chromosomes into smaller fragments that can be easily manipulated.
*Copy mRNA into DNA and insert into a plasmid for analysis
*Expression of protein

Front 23

HOW DO YOU VISUALIZE DNA?

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USE ELECTROPHORESIS
NOTES
•DNA fragments migrate in electric field
•Larger ones will move SLOWER
•Fragments of same mass will migrate to same location
•Must visualize DNA with dye or radioactivity

ETHIDIUM BROMIDE
Dye that intercalates into the DNA double helix and fluoresces under UV light
HIGHLY TOXIC
MUTAGEN

Front 24

Brief history of GFP:

Back 24

GFP gene cloned in 1992
Jellyfish (Aequoria victoria)
First fluorescent protein to be isolated

Front 25

Key properties of GFP are:

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Absorption and emission wavelengths of the protein are in the visible region
(>350nm) (ie. We can see it)

Fluorescence is autocatalytic and instrinsic to the protein
(ie. It does not require any cofactors that are not already in cells)

The GFP can be expressed in ANY cell or organism and can be observed in a LIVING cell (ie. The cell does not have to be fixed and broken open to observe the location)

Front 26

Wild type GFP is this size:

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238 amino acids
27kDa

Front 27

Fluorescence is generated by the cyclization and oxidation of the sequence:

Back 27

S-Y-G

Front 28

GFP is very stable due to the fact that:

Back 28

it forms a cylinder of beta sheets

Front 29

In GFP this is protected in the center of the beta sheet cylinder

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S-Y-G sequence

Front 30

GFP excitation/emission wavelengths:

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Excitation = 475 nm
Emission = 509 nm

Front 31

BASIC STRUCTURE OF ANTIBODY:

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2 heavy chains
2 light chains

Fab region is for antigen binding
Fc region is effector domain
- signal to body that Ab has bound

Front 32

The antibody will bind with high affinity to:

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only one protein

Front 33

BASICS OF WESTERN BLOTTING

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Blotting
Blocking
Primary staining
Secondary staining
detection

Front 34

In blocking:

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• Need to mask reactive sites
• Consider concentration of the protein you want to detect vs all the other
proteins
• Need something that will not react with the Ab

Dry milk is used. Albumen will coat the blot, low binding to antibodies. Signal to noise

Front 35

Primary Antibody against target (EYFP):

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– Produced in rabbits
– Used at 1:100 dilution
– Incubated with the blot

Front 36

Secondary Antibody:

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• Is an antibody that will bind to the primary
• Is covalently conjugated to an enzyme
– Alkaline Phosphatase
– Used at 1:5000

Front 37

This is used in detection in the western blot:

Back 37

NBT
BCIP
Alkaline phosphatase will metabolize
-blue precipitate