Download MODERN METHODS in BIOCHEMISTRY

MODERN METHODS in
BIOCHEMISTRY
•PROTEIN MODIFICATION
•PROTEIN CROSSLINKING
•PROTEIN STAINING
•ANTIBODY MODIFICATION
•IMMUNOPRECIPITATION
•METABOLIC LABELLING
Affinity chromatography :
• matrices – preparation
• Coupling of Ligand
• detection of reactive groups
Affinity chromatography :
Principle of method
Affinity chromatography
Mouse brain
Example
Affinity Chromatography
Immunoprecipitation
Gel filtration
Solubilise & homogenise
50,000 x g
supernatant
eluted complexes
SDS-PAGE
Affinity
chromatography
matrice preparation
& introduce spacer
Affinity chromatography :
Coupling of Ligand
Affinity chromatography Example
COMMONLY USED AGENTS
1. Cyanogen bromide
• Cyanogen bromide (CNBr) is one of the most widely
used linkers in affinity chromatography. It reacts with
hydroxyl groups in agarose and other polysaccharide
materices to produce a reactive linker. The linker can
be connected to ligands or spacers which contain
primary amine groups (such as proteins).
• Cyanogen bromide is extremely toxic as it releases
hydrogen cyanide on acidification.
COMMONLY USED AGENTS
2. Bis-Expoxides (Bis-oxirane)
• Bis epoxides react readily with both
hydroxyl or amino-containing gels at
alkaline pH.
• This produces a long chain hydrophilic
reactive oxirane. This can then be
linked to ligands.
• The long chain oxirane itself acts as a
spacer.
COMMONLY USED AGENTS
3. Carbonyldiimidazole
• N,N'carbonyldiimidazole (CDI) is reacts with
polysaccharides to form imidazole carbonate
derivatives. These derivatives then react with ligands
containing primary amino groups at an alkine pH to
give stable carbonate derivatives.
4. Sulphonyl chloride
•
Reacts with hydroxyl groups on the matrix to
form sulphonyl esters. These esters react with amino
and thiol groups of the ligand.
5. Sodium periodate (NaIO4)
•
Reacts with diol groups on polysaccharide matrices to
form reactive aldehydes. These react with primary amines
to form Schiff's bases.The Schiff's bases can be stabilized
by reduction with sodium borohydride.
•
Periodate is easy to use and non-toxic. The product is
stable.
6. Glutaraldehyde
• Activates amino or amide groups on polyacrylamide
matrices or on agarose with amine spacers. These groups
can then react with primary amine groups on the ligand.
Tags and Fusion sequences
•
Many vectors are now engineered with DNA sequences encoding a specific
peptide (purification tag) that, when fused to the expressed protein, may be
used for one-step purification of the recombinant protein by high-affinity
binding. That is molecular biologists design expression systems such that the
recombinant protein can be easily purified using affinity chromatography.
•
The technique involves linking the gene coding for tag to that coding
for the protein of choice. When the latter is expressed, the resulting tag
is also produced and is linked to the desired protein. The tag is
selected such that it can be readily be bound a particular affinity
chromatography material.
• Example 1 : 6xHis system : six histidine residues are tagged to
the recombinant protein. The histidine tag and protein can be purified using
a nickel-chelating resin.
• Example 2 : pMal system : tags = maltose binding protein to the
recombinant protein. The maltose binding protein tag will bind strongly to an
amylose column.
Brand Name
Ligand
Application Examples
HiTrap Protein A
Protein A
IgG, IgG subclasses, fragments of
IgG, IgA, antigens, immune
complexes
HiTrap Progein G
Recombinant Protein G
IgG, IgG subclasses
lacking the albumin binding region
HiTrap Heparin
Heparin
Growth factors, coagulation proteins,
plasma proteins, lipases, lipoproteins,
enzymes that act on nucleic acids,
steroid receptors, protein synthesis
factors
HiTrap Chelating
Iminodiacetic acid
Proteins and peptides with exposed
histidine groups
HiTrap Blue
Cibacron Blue F3GA
Enzymes requiring adenyl-containing
cofactors, albumin, coagulation
factors, interferon
Brand Name
Ligand
Affi-Gel Protein A
Affi-Prep Protein A
Protein A
Applications
Purification of IgG from acites, serum and
culture fluid.
Affi-Gel Blue
Cibacron Blue F3GA
Binds many nucleotide-requiring enzymes,
albumin and other proteins.
DEAE Affi-Gel Blue
Cibacron Blue F3GA
and DEAE groups.
Binds albumin and serum proteins
Used to purify protease-free IgG from acites,
serum and cutlure fluid
Affi-Gel heparin
Heparin
Purification of a wide variety of proteins
including growth factors, coagulation factors,
DNA and RNA specific enzymes, lipase,
lipoproteins and proteases
Affi-Prep polymyxin
Polymixin
Endotoxin removal
Affi-Gel 501
Organomercurial
Adsorbtion of sulfhydrl proteins and low mw
sulfhydrls via thiol groups. Bound proteins are
eluted with dilute mercaptoethanol or dithioreitol.
Affi-Gel 601
Boronate
Adsorbtion of cis-hyroxyl containing molecules
including sugars, nucleotides and glycocpeptides.
MODERN METHODS in
BIOCHEMISTRY
•PROTEIN MODIFICATION
•PROTEIN CROSSLINKING
•PROTEIN STAINING
•ANTIBODY MODIFICATION
•IMMUNOPRECIPITATION
•METABOLIC LABELLING
PROTEIN STAINING
PROTEIN STAINING
• Amidoblack
• Commassie
• Ponceau-red
• Silver
• Gold
• Gelcode
Protein Imaging
Coomassie Blue Dyes
- commonly used
- does not interfere with subsequent protein identification
- inexpensive
- sensitivity well below silver and fluorescent dyes
Silver stain
- sensitivity 10-50 times greater than CB
- ability to detect 1 ng of protein
- silver diammine/silver nitrate
- relatively expensive (reagents/waste disposal)
- high background
Fluorescent Stains and Dyes
- accurately determine changes in protein expression
- greater sensitivity than silver stain
- DIGE
- cost
Silver-stained two-dimensional polyacrylamide-gel
PROTEIN STAINING
Amidoblack
spot 5 ul protein sample on nitrocellulose (NC)
- air-dry for 5 min
- immerse in stain solution for 3 min
-
wash 2x 3 min in water
wash 2x 3 min in wash solution
wash 5 min with water
air-dry 5 min
- elute stain with 1 ml elution solution (while shaking the sample)
- measure absorption at 630 nm of the eluant
PROTEIN STAINING
Amidoblack
stain = 0.1 % amidoblack, 45% methanol, 10% acetic acid
wash = 90% methanol, 2% acetic acid
elution = 50% ethanol, 50% 50 mM NaOH/0.1 mM EDTA
- use BSA solutions (0 to 5 mg/ml) for calibration
- this protocol also works for proteins in SDS-PAGE buffer
PROTEIN STAINING
Coomassie Blue
•
1. Immerse gel in 50% ethanol/10% acetic acid for at least 1 hr.
•
2. Soak in 5% ethanol/5% acetic acid overnight or for a
minimum of 2 hours.
•
3. Wash in diH2O for 1 hr.
•
4. Add Gel-Code Blue Stain reagent for at least 3 hrs.
•
•
•
(Pierce, #24592)
5. Wash in diH2O twice, 15 min each.
6. Rinse in diH2O for 1 hr.
7. Gels can be stored at 4°C.
PROTEIN STAINING
Coomassie-Blue
&Amidoblack
PROTEIN STAINING
Ponceau Red
• For Membrane after Blotting. To check blotting
• Reversible: gets washed out with destaining
solution
•
Ponceau S stock solution: 200 mg Ponceau S per
100 ml 3 % Perchloric acid
• ready-to-use solution : dilute stock sol. 1:5 with
10% acetic acid
•
Background destaining :10 %acetic acid
PROTEIN STAINING
Silver
PROTEIN
STAINING
OTHER STAINS
Gold-Stain
Gold-Blot
Silver-Stain
Sypro-Orange
BioRad-Zinc
Coomassie-Blue
Sypro-Red
MODERN METHODS in
BIOCHEMISTRY
•PROTEIN MODIFICATION
•PROTEIN CROSSLINKING
•PROTEIN STAINING
•ANTIBODY MODIFICATION
•IMMUNOPRECIPITATION
•METABOLIC LABELLING
IMMUNOPRECIPITATION
IMMUNOPRECIPITATION
APPLICATIONS-I
Immunoprecipitation can be used for many purposes :
• 1) Determination of the molecular weight and
isoelectric point of immunoprecipitated proteins by
one-dimensional or two-dimensional SDS-PAGE.
• 2) Verification that an antigen of interest is
synthesized by a specific tissue (i.e., that radiolabeled
protein can be identified in tissues or cells cultured
with radiolabeled precursors).
• 3) Determination of whether a protein contains
carbohydrate residues by evaluating whether
immunoprecipitated antigen from cells cultured with
radioactive monosaccharides is radiolabeled.
IMMUNOPRECIPITATION
APPLICATIONS - II
• 4) Characterization of the type of carbohydrate
present on glycoproteins - evaluate incorporation of
different radiolabeled monosaccharides into
immunoprecipitated protein during cell culture and test
whether inhibitors of glycosylation alter the molecular
weight of immunoprecipitated protein.
• 5) Determination of precursor-product relationships by
performing pulse-chase labeling followed by
immunoprecipitation.
• 6) Quantification of synthesis rates of proteins in
culture by determining the quantity of
immunoprecipitated, radiolabeled protein.
Immunoprecipitation
Mixed
proteins
Agarose bead conjugated
to secondary antibody
Add
antibody
Add
beaded
αbody
= HA-VEGFR
(Haemagglutinin)
resuspend pellet
and load on gel
IP - anti HA
probe - α p-VEGFR
Spin
Immuno-co-precipitation
= shc
Primary
Secondary
(mouse α-HA) (rabbit α-mouse)
IP - anti HA
probe - anti shc
Primary sheep α Shc
Secondary donkey α sheep
VEGF
IP anti HA
VEGF
IP anti HA
Warner et al Biochem. J. (2000) 347, 501–509
Kinase assays
1. Add radioactive phosphate
and substrate
2. Subject to SDS PAGE
3. Develop against film
32P
32P
32P
labelled
substrate
Unincorporated
32P
Porcine aortic endothelial cells tranfected with VEGF-R2
PI3 Kinase inhibitors
Vehicle
Stimulant
Qi & Claessen-Welsh, Exp Cell Res 263 173-182 (2001)
IMMUNOPRECIPITATION
Explants cultured in the presence of
[35S]met incubated w. Ab to uterine milk
protein (UTMP).
Ag-Ab absorbed w. Protein A-Sepharose
SDS-PAGE + fluorography.
Ab = proteins immunoprecipitated with
rabbit antiserum to UTMP
NRS = normal rabbit serum
TC = total array of radiolabeled proteins
present in the unabsorbed sample
IMMUNOPRECIPITATION
SOME PROBLEMS I
• 1) crossreactivity : attention must be given to
antibody cross-reactivity with other antigens (like all
immunochemical procedures)
• 2) Nonspecific binding : can be a problem
especially if proteins that are immunologically distinct
from the antigen are trapped in the pellets formed
during immunoprecipitation.
IMMUNOPRECIPITATION
• To reduce nonspecific binding, immuno-precipitation
buffers usually have
– some detergent to reduce hydrophobic interactions,
– a protein to block nonspecific binding sites,
– and high salt to reduce ionic interactions.
• In many protocols, a preclearing step is performed
to remove molecules that nonspecifically bind to
insoluble Protein A or Protein G.
• Despite these precautions, nonspecific binding can
occur.
IMMUNOPRECIPITATION
It is crucial, therefore, to always perform a control
reaction where antibody is replaced by a non-relevant
immunoglobulin (i.e, normal serum for polyclonal
antibodies, control mouse ascites fluid for ascites, and
isotype controls for purified mouse monoclonal
antibodies).
IMMUNOPRECIPITATION
SOME PROBLEMS - II
• 3) Proteolytic digestion : can occur when cells are lysed
and contents of lysosomes are mixed with other compartments
of the cell.
Accordingly, most immunoprecipitation buffers contain
proteinase inhibitors.
• 4) Care should be taken in using immunoprecipitation as a
quantitative tool to determine rates of synthesis of proteins
because the rate of incorporation of radiolabel into protein will
depend upon rate of synthesis of a protein as well as the rate of
dilution of radiolabeled precursor by the intercellular pool of
precursor.
IMMUNOPRECIPITATION
SOME PROBLEMS - III
• 5) Protein Trapping : molecules too small or too large to be
resolved by SDS-PAGE are sometimes trapped in the pellet
formed by immobilized Protein A or Protein G.
•
These molecules, while not interfering with analysis by SDS-PAGE, can
make direct quantification of radiolabeled antigen by scintillation
spectrometry problematic.
• Thus, immunoprecipitated protein should be quantified by
densitometric analysis of autoradiographs or fluorographs.
Scintillation spectrometry sometimes reveals little difference in the
quantitative yield of radioactivity between an immunoprecipitation
reaction and a control reaction (i.e., where antibody has been
substituted with normal rabbit serum). Nonetheless, subsequent
analysis by SDS-PAGE reveals precipitation of radiolabeled protein in
the antibody reaction only.
IMMUNOPRECIPITATION
SOME PROBLEMS - IV
• 6) Sensitivity : can be a problem,
especially when the antigen is a minor component of the
protein pool.
New screen technology for low energy radioisotopes (Transcreen
LE enhancing screens by Kodak) increases sensitivity greatly.
Use as much protein in the immunoprecipitation reaction as
possible.
III. FASPS (4)
Serine to glycine mutation in hPER2 of FASP person
Fig 13. Mapping of the CKIε binding domain of PEFig 14. In vitro CKIe phosphorylation of wild
R2 by immunoprecipitation(Toh et al, 2001).
type and mutant hPER2(Toh et al, 2001).
anti-MYC
antibody
mper2 cDNA
PER2
myc
PER2
CKIε
rabbit
reticulocyte
ck1ε cDNA
myc
CKIε
Immuno-precip
itation
Separation in SDS P
AGE gels
<Experimental scheme>
-8-
MODERN METHODS in
BIOCHEMISTRY
•PROTEIN MODIFICATION
•PROTEIN CROSSLINKING
•PROTEIN STAINING
•ANTIBODY MODIFICATION
•IMMUNOPRECIPITATION
•METABOLIC LABELLING
METABOLIC LABELLING
• Aims:
METABOLIC LABELLING
• Aims
: An important measurement of
metabolism in metabolic engineering is
in vivo metabolic flux: the rate of flow of
biochemical material down a pathway.
A
B
C
D
Rates expressed in units of quantity for unit time per unit tissue mass e.g. nmol.min
nmol.min-1.g-1 Fw
Metabolic Flux Analysis (MFA)
Stoichiometric flux balance analysis
Isotopic flux balance analysis :
steady-state
steady-state
radiolabeling
non-steady-state
non-steady-state
dynamic
kinetic
models
stable isotope labeling
steady-state
All types of MFA involve algebraic representations
of metabolism with numerous interacting variables. Effective handling of
these interacting variables requires computer simulation models.
Principles of flux determination from
radiolabeling kinetics
(external radiolabeled M)
M*
J
M
J
The flux, J, can be determined by introducing a
pulse of radiolabeled M (M*) and measuring the
radioactivity in samples of purified M.
The pool of intracellular radiolabeled
M will change according to the
following equation:
dM*
M*
dt
M
J
=Stephanopoulos, G.N., Aristidou, A., Nielsen, J. 1998. “Metabolic Engineering
Principles and Methodologies”. Academic Press, San Diego.
Radiolabeling Experiments
14C, 33P-labeled
precursor
Leaf disk labeling
1. Labeled metabolite of known
specific activity supplied
2. Extraction of leaf tissue
3. Phase separation
4. Ion exchange chromatography
5. Thin layer chromatography
or electrophoresis
6. Quantify metabolites
by scintillation counting
Practical concerns
Is the supplied labeled compound taken up?
Is it altered before uptake ? (e.g., phosphorylated compounds)
Can you separate all relevant metabolites?
Are the metabolites efficiently recoverable?
Advantages of Radiolabeling
Experiments
• Very high sensitivity
- can quantify turnover in small pools
- only small tissue quantities are required; replication,
many
time points possible fore kinetic studies
• Straightforward quantification (scintillation counting)
- must account for quenching (pigments, solvents)
- metabolites must be separable so that label in different
compounds not counted together.
- metabolites not labeled via intersecting pathways
- need to consider the potential for labeling of multiple
atoms
• High time resolution
- critical for rapid kinetics
METABOLIC LABELLING
How long should cells be labeled?
• The ideal length of time to label cells depends on the protein of
interest and the label that you are using.
• If you want to label an unstable protein with 35S-methionine, a
short labeling interval--no more than 2 hr—is best.
• If you are studying a stable protein, a longer label may be
preferable.
• The issue is the half-life of the protein of interest relative to the
half-life of the background bands. The half-life of total cellular
protein is 45 to 50 hr.
METABOLIC LABELLING
Labeling with 32Pi is different.
• the phosphate in proteins undergoes continual turn-over in
most cases. Therefore, both old and newly-synthesized
proteins become labeled soon after 32Pi is added to the cells.
• A short labeling period with 32Pi is advantageous :
labeling of RNA and DNA is less obvious than in cells labeled over-night.
• For studying tyrosine protein kinases: phosphotyrosines tend to
turn-over faster than the bulk of either phosphoserine or phosphothreonine
and thus are preferentially labeled during brief labeling.
METABOLIC LABELLING
• over-night labeling period is optimal if looking at
the steady-state abundance of phosphate in proteins, lipids, or
RNA,
• Under these conditions, the specific activity of the ATP pool in
the cell and of the phosphates in macromolecules is beginning
to approach that of the medium.
• Additionally, the amount of label present in proteins, lipids and
RNA should now reflect the amount of phosphate present,
rather than the rate of turn-over of the phosphate.
METABOLIC LABELLING
• An issue to consider : radiation damage !!!
• Can induce the stabilization of p53 and cause cell cycle arrest.
• Cells labeled for a prolonged period with 32P may therefore not
be growing when you harvest them.
• the specific activity of the ATP pool comes to equilibrium with
the phosphate in the medium by 6 hours. Therefore there is no
reason to label for longer with 32Pi.
METABOLIC LABELLING
• Overnight labeling is best done in medium containing a
reduced concentration of phosphate or methionine. 10% is
often reasonable, depending on the cell line.
• easily accomplished by using methionine-free or phosphatefree medium and 10% undialyzed serum (which can be assumed to
contain the same concentration of methionine or phosphate as normal medium).
METABOLIC LABELLING
• For short term labeling (30 seconds to 5 hrs)
Use :
•
•
•
•
(1) medium completely lacking either phosphate or methionine,
(2) serum which has been dialyzed against saline, and
(3) a fairly low volume of medium;
0.75 ml for a 35 mm dish, 2 to 2.5 ml for a 50 mm dish, and 2.5 to 5 ml
for a 100 mm dish.
• It is a good idea to rinse the cells you are going to label with
labeling medium—which lacks label—prior to adding the actual
labeling medium.
• Starvation doesn't help much.