Reviewer For LBYBIOJ MIDTERMS

Standard Curve Preparation for

Determining Protein Content

concentration of the substance and the path

length of the light through the solution.

Cell Fractionation and Separation

Protein Quantitation isolation,

characterization, purification, and
identification

Homogenization, to break open cells to

separate their structural and molecular
components.

Procedures that need qunatified protein

samples:
chromatography
electrophoresis
functional assays
immunochemical separation &
analyses

Homogenization Methods
Detergents (SDS)
Salts for Osmotic Alteration
Enzymes (trypsin and proteinase K)
Mechanical methods
Ultrasonification (sound waves)

280 nm, abs of protein molecules in solutions

due to presence of aromatic amino acids
(tyr & trp)
Zero Buffer Absorbance, to resolve
interference of substance in the buffer for
absorbance.

Fractionation, separation or subcellular

organelles by centrifugation (by density)
Pellet material that collects at bottom of
microfuge tube
Supernatant fluid above the pellet

Crude, homogenized material, all

Colorimetric Methods of Quantitation
Protein dye-binding Chemistry
(Coomassie/Bradford)
BCA Protein Assay
Modified Lowry Protein Assay
Protein-copper Chelation Chemistry
Selection of Protein Standard
h i g h l y p u r i fi e d v e r s i o n o f m o s t
predominant protein in sample
Bovine Serum Albumin (BSA)
Biuret Assay
Biuret, small compound formed when
urea is heated causing two urea
molecules to join. Copper complexes
produced produce strong blue color.
Bradford Assay
Coomassie Brilliant Blue G-250
binds to basic (especially Arg) and
aromatic amino acid residues
Cationic (Red) [470 nm]
Neutral (Green)
Anionic (Blue) [595 nm]
Beers Law, the quantity of light absorbed by
a substance dissolved in a fully transmitting
solvent is directly proportional to the

components present
Nuclei (pellet)
Soluble (supernatant)
Microsome (pellet)
595 nm, abs to be read at

Marker Enzyme Assay

Marker Enzymes enzymes localized in a
particular organelle of the cell
A. Alkaline Phosphodiesterase (APDE)
Acid
present in phagocytic vacuoles
thymidine 5 monoP > p-nitrophenol
yellow compound

Buffer D (Tris-borate, Triton X-100,

MgCl2, ZnSO4)

Substrate D (p-nitrophenyl thymidine 5

monophosphate)

B. Peroxidase Assay
present in peroxisomes
Peroxidase oxidized reduced TMB in
presence of H2O2
Blue color

SDS - Sodium Dodecyl Sulfate

gives proteins net negative charge
removes 2ndary and 3rtiary structures
Ammonium Persulfate and TEMED
POISONOUS

Tetramethyl Benzidine (TMB)

C. Acid Phosphatase Assay

Present in Lysosomes
pNPP or pNTP > para-nitrophenol
Yellow Compound
Buffer C (glycine-HCl, Triton X-100)
Substrate C (p-nitrophenol phosphate)
D. Mitochondrial Reductase/
Dehydrogenase Assay
Reduction of Blue Resazurin > Red
Resofurin

Alamar Blue Viability Reagent

E. Protease Acitvity Assay

Protease, may connote some physiological

conditions such as stress
Differential Scanning Fluorimetry (DSF)
Proteases digest BSA, products bind to
Flamingo > enhanced fluorescence
Relative Fluorescence Units (RFU),
measure of fluorescence

BSA or Chicken Egg White Albumin

Flamingo Pink Fluorescent Stain
DNAse key enzymes released during
apoptosis, responsible for damaging DNA
SYBR Green dramatic fluorescence in
presence of double stranded DNA

SDS-Polyacrylamide Gel
Electrophoresis
Gel Electrophoresis separates charged
molecules by running through a matrix (gel)
in an electrical current.
Nucleic Acids net negative charge due to
their phosphate group
Cathode (NEGATIVE), Anode (POSITIVE)

Mini-Protean III vertical slab unit designed

for faster electrophoresis

4% Acrylamide Stacking Gel, restricts

protein migration, concentrate and properly
align protein samples
12% Acrylamide Running Gel,
separates individual polypeptides into
discrete bands
Staining
Coomassie Blue R250
SYPRO Ruby

SDS-PAGE Gel Image Analysis

Protein Profiling data from SDS-PAGE
used to determine how closely related two or
more species are at the level of expressed
gene products

Protein Sequence Analysis and

Homology Modeling of 3D
Structures
Reverse Genetics Once the gene
sequence is known, the exact amino acid
sequence can be deciphered and thus will
show more the inherent properties of the
protein being investigated.
Amino Acid = Residue
Amino Acid alpha carbon, carboxylate
group, amino group, R group (gives property)
Affects Structure of the Protein
e.g proline, chain breaker because of
its cyclic structure
glycine, mabilis magfold lol
Primary Structure - amino acid sequence
Secondary Structure structure, folding

Types of Secondary Structures

1.alpha helix glycine on turn position,
3.6/3.4 amino acid residue per turn.
Maintained by Hydrogen bonding
2. beta sheath
3. Random coil randomly
coiled, still a type of secondary
structure
4. Turn not necessarily for the alpha helix
Tertiary Structure 3d
Quaternary Structure - subunits
Dimeric, trimeric, multimeric, etc.
Xray Crystallography/Protein
Crystallography determining 3d
protein structure
**you need to have a protein signal
Crystal ordered periodic material
**more reliable than NMR
NMR Nuclear Magnetic Resonance
Concept: Poly exclusion principle
Spots correspond to the
structure of the protein
Downside: difficult to interpret
*like taking a video of the
protein, measuring the structure in
solution.
Why bother?
Structure-function relationship
Homology Modelling
- Refers to the process wherein the
structure of the protein is
predicted based on a template
- The template must be well-known,
a crystal structure (?)
- Structures are conserved within
the family of the proteins
evolution
Ramachandran Plot
- Plots phi and psi angles,
corresponds to the tortional angles
of the peptides
o Alpha carbon and the
nitrogen
Tortional angle between
alpha carbon and nitrogen
Psi angle between carbonyl
carbon and alpha carbon

Omega Angle not included

in the ramchandran plot
Between carbonyl
carbon and
nitrogen amide
bond
It does not give you
any idea about the
protein structure
Becausde of its
rigidity (partial
double bond
character), hindi
masyadong
nakakaikot