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Western blotting
To detect the antibody use a secondary antibody against the primary antibody (e.g, goat anti-rabbit IgG).
The secondary antibody is a commercial fusion protein with an enzyme activity (e.g., alkaline phosphatase).
The enzyme activity is detected by its catalysis of a reaction producing a luminescent compound.
http://www.bio.davidson.edu/courses/genomics/method/Westernblot.html
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Last updated Nov. 15, 2011 5:11 PM
2
Y YNon-luminescent substrate
Luminescent product
Protein band on membrane
Horseradish peroxisase fusion protein, e.g.
Secondary antibody(e.g., rabbit anti-mouse IgG)
Antibody to protein on membrane
Detect by exposing to film(minutes or hours). Can be quantitative.
Detection of antibody binding in western blots
(chemiluminescence)
Horseradish Peroxidase (HRP)
Luminol
ECl = extended chemiluminescence
3
WB = western blot
FLAG and Myc are epitopes for which there are good antibodies available.
GST = glutathione-S-transferasePABP2 = PolyA binding protein 2
RRMs = PABP2 RNA recognition motif
PABP2-FL full length proteinPABP2-N N-terminal fragment
Western blotting
Myc-SKIP = SKIP protein with a myc tag
Pulldown result
Reagentswork
antiPABP2 co-IPsSKIP
Ig H-chain
Co-tranfect.
4Far western blotting to detect specific protein-protein interactions. Use a specific purified protein as a probe instead of the primary antibody
To detect the protein probe use an antibody against it.
Then a secondary antibody against the first antibody, a fusion protein with an enzyme activity.
The enzyme activity is detected by its catalysis of a reaction producing a luminescent compound.
http://www.bio.davidson.edu/courses/genomics/method/Westernblot.html
protein protein
OR:Use a radioactively labeledprotein of interest and detect by autoradiography
5How to make a radioactively labeled protein:Expression via in vitro transcription followed by in vitro translation
cDNA
T7 RNA polymerasebinding site (17-21 nt)
….ACCATGG…..
VECTOR
2. Add a translation system: rabbit reticulocyte lysate or wheat germ lysate
Or: E. coli lysate (combined transcription + translation, TnT)
All commercially available as kits
Add ATP, GTP, tRNAs, amino acids, label (35S-met), May need to add RNase (Ca++-dependent, stop with EGTA) to remove endogenous mRNA In lysate
1. Transcription to mRNA via the T7 promoter + T7 polymerase
Radioactively labeled protein
NOTE: Protein is NOT at all pure (1000s of lysate proteins present), just ~“radio-pure”
6Surface plasmon resonance (SPR)Popular instrument is a Biacore
The binding events are monitored in real-time and it is not necessary to label the interacting biomolecules.
http://home.hccnet.nl/ja.marquart/BasicSPR/BasicSpr01.htm
glass plate
Reflection angle changes depending on the mass of the material on the surface.Binding increases this mass. Follow as a function of concentration Kd’sOr time : Measure on-time, off time; Kd = off-time/on-time
In a flow cell
7
A Biacore result
Ligand added
Ligand removed
8
SW Michnick web site: http://michnick.bcm.umontreal.ca/research/images/pca_general_en.gif
F = reporter protein fragment
Enzyme fragmentsthemselves do not associate well enough to reconstitute an active enzyme
Reporterenzyme
Back to protein-protein interactions:
9
SW Michnick web site: http://michnick.bcm.umontreal.ca/research/images/pca_general_en.gif
F = reporter protein fragment
Enzyme fragmentsthemselves do not associate well enough to reconstitute an active enzyme
Reporterenzyme
Back to protein-protein interactions:
10Folic acid
DHFR
DHFR
http://www.nature.com/onc/journal/v22/n47/images/1206946f1.gif
(FH4)
(FH2)
Dihydrofolate reductase (DHFR): role in metabolism
11
FK506 = immunosuppressant drugFKBP = FK506 binding proteinFRAP = FKBP12–rapamycin associated proteinFRB= FKBP–rapamycin binding domain of FRAP
DHFR = dihydrofolate reductaseDHF=dihydrofolate = FH2
THF=tetrahydrofolate = FH4
fMTX=fluorescent methotrexate
fMTX
DHFR fragments
Clonal selection and in vivo quantitation of protein interactions with protein-fragment complementation assays, I. Remy and S.W. Michnick PNAS 96, 394–5399, 1999
IN PURINE-FREE MEDIUM
Fluorescein – MTXbinding assay
Rapamycin promotes the association of the 2 protein domains
Cell growth assay: CHO
DHFR- mutantcells
12
a phosphatase
FK506 recruits FKBP to bind to calcineurin and inhibit its action as a specific phosphatase
13
Claim detection of 0.05 nM rapamycin??
No.
of
CH
O c
olon
ies
[rapamycin]
14
Background association of FKBP and FRB without rapamycin
(compare mixed input)
Leucine zipper protein fragments instead ofrapamycin binding proteins (positive contro)
CHO cells(permanent transfection)
cos cells(transient transfection)
Fluorescent methotrexate (fMTX) assay:Wash in, wash out
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8-fold increase in fluorescence per cell
Measure affinity for a drug in vivo
Fuorescence-activated flow cytometer(FACS is this, plus more)Allows quantitation of fluorescence per cell
No.
of
cells
Flu
ores
cenc
e in
tens
ity
Log of fluorescence intensity
[rapamycin]
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EMP1 = Erythropoietin mimetic peptide 1
Another domain-domain interaction measured:Erythropoietin-erythropoietin receptor (dimer) interaction: Efficacy of a peptide mimetic
Erythropoietin
In vivo assay of drug effectiveness (EMP1)(inexpensive substitute for erythropoietin?)
Erytropoietin (EPO) receptor
EPO bp1EPO bp2EPO
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FACS = Fluorescence-activated cell sorter
Impart a charge on the recognized cell
Less than one cell or particle per droplet. Thus the most that most droplets contain is one particle.
Charged plates attract droplets containing a particle of the opposite charge
Cells remain viable if treated with care.
Can be used purely analytically without the sorting capability. Thencalled “flow cytometry”, or also called FACS anyway.
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No.
of
cells
Having this much fluorescence
Histogram-type display
No fluorescence (background autofluorescence)
Red stained
Usually a log scale
19
One cell
Amount of red fluorescence (log)
Am
ount
of
gre
en f
luor
esce
nce
(log)
Say, want high reds butlow greens:Instruct the FACS to deflect cells in this quadrant only. Collect and grow or analyze further.
Analysis on 2 colors
Scatter plot display
You decide on the positions of of demarcations
Rice paper cont.
TPA = Tissue plasminogen activator, dissolves clots
Problem: Cleared quickly from bloodstream by liver
Bind to hepatocytes in liver via TPA’s kringle domain
Want to isolate a TPA mutant protein with less affinity for hepatocytes
Must be still enzymatically active of course.
Goal: to improve tissue plasminogen activator as a therapeutic “clot-busting” treatmentMeans: Reduce or eiminate the binding of tPA to liver cells, as this clears it from the blood
Authors here use a mammalian cells as the carrier of the DNA and the cell surface as a display site. Display was via a fusion protein to a membrane anchor protein, DAF (peptide, really).DAF = “decay accelerating factor”
What did they do?Cassette mutagenesis.
What region?333 bp K1 (kringle-1), known to bind the MAb387, which competes for hepatocyte binding (so assuming it is the same target epitope).
How did they get kringle mutated?Error-prone PCR
How did they isolate just the kringle 1 region? PCR fragment.
How did they get the mutagenized fragment back in?Introduced restriction sites at the ends, w/o affecting the coding.
What did they put the mutagenized fragment into?DAF – TPA fusion protein gene
How did they get it into into cells?Electroporation
What cells did they use as hosts?293 carrying SV40 large T antigen
How many copies per cell. And why is that important? One, by electroporation at low DNA concentration. [In a transient transfection!]Binding is dominant. Lack of binding (what they are after) is recessive.
How did they select cells making MAb387-non-binding TPA?FACS:Recover cells that bind fluorescent mAb vs. protease domainbut low binding to fluorescent mAb vs. kringle domain
Tracked down vector: contains SV40 ori and is transfected into 293 cells making SV40 T-antigen. So plasmid replicates during the transient transfection higher signal.
For reiteration of the process
Sort the cells with low fluorescence
,
How did they recover the plasmid carrying the mutant TPA gene from the selected cells?
Hirt extraction: Like a plasmid prep, lyse cells gently, high MW DNA entangles and forms a “clot”.Centrifuge. Chromosomal DNA soft pellet; plasmid DNA circles stay in supernatant.
Then re-transfect, re-sort in FACS.
After 2 sorting rounds, test individual E. coli clones: 60% are binding-negative.
MAb to protease domain
MAb to kringle-1 domain
enriched
Low kringle-1 reactivity
FITC = fluorescein reagent. PE = phycoerythrin (fluorescent protein)
No good
good good good good
Collect these
Log plots
Hepatoma cell binding. How?
Clone mutated regions into regular TPA gene for testing (no DAF, protein now secreted)
Label WT TPA with fluorescein (FITC, conjugated chemically) Mix with hepatoma cells and analyze on a flow cytometer (FACS w/o the sorter part).
See specific and non-specific binding. Subtract non-specific binding: the amount not competed by excess un-labeled wt TPA.
FITC = fluorescein isothiocyanate
WT
Compete. So still bind.
But still haveprotease activity
Hepatoma cell binding assay:measure competition for binding of fluorescently labeled WT TPA
Can’t compete (good)No competitor
Binding assay, initial condition
31
Got this far
Mammalian cell geneticsIntroduction:
Genetics as a subject (genetic processes that go on in somatic cells: that replicate, transmit, recombine, and express genes)
Genetics as a tool. Most useful the less you know about a process.
4 manipulations of genetics:
1- Mutation: in vivo (chance + selection, usually); targeted gene knock-out or
alteration in vitro: site directed or random cassette
2- Mapping: Organismic mating segregation, recombination (e.g., transgenic mice);
Cell culture: cell fusion + segregation; radiation hybrids; FISH
3- Gene juxtaposition (complementation): Organisms: matings phenotypes of heterozygotes; Cell culture: cell fusion heterokaryons or hybrid cells
4- Gene transfer: transfection
Mammalian cell geneticsAdvantages of cultured cells (vs. whole organism): numbers, homogeneity
Disadvantages of cultured mammalian cells: limited phenotypeslimited differentiation in culture (but some phenotypes available) no sex (cf. yeast)
Mammalian cell lines
Most genetic manipulations use permanent lines,for the ability to do multiple clonings
Primary, secondary cultures, passages, senescence.Crisis, established cell lines, immortality vs. unregulated growth.
Most permanent lines = immortalized, plus "transformed“, (plus have abnormal karyotypes)
Mutation in cultured mammalian cells:
Problem of epigenetic change: Variants vs. mutants
Variants could be due to:Stable heritable alterations in phenotype that are not due to mutations:
heritable switches in gene regulation (we don’t yet understand this).
DNA CpG methylation, histone acetylation / de-acetylation
Diploidy. Heteroploidy. Haploidy.
The problem of diploidy and heteroploidy: Recessive mutations (most knock outs) are masked.(cf. e.g., yeast, or C. elegans, Dros., mice): F2 homozygotes)
Solutions to diploidy problem:Double mutants (incl. also mutation + segregation, or mutation + homozygosis:(rare but does occur)
Heavy mutagenesis, mutants/survivor increases but mutants/ml decreases.
How hard is it to get mutants? What are the spontaneous and induced mutation rates? (loss of function mutants)Spont: ~ 10-7/cell-generation Induced: ~ 2 x 10-4 to 10-3 /cell (EMS, UV)
So double knockout could be 0.00072~ 5X10-7. One 10cm tissue culture dish holds ~ 5x106 cells.
Note: Same considerations for creation of recessive tumor suppressor genes in cancer: requires a double knockout. But there are lots of cells in a human tissue or in a mouse.
RNAi screen, should knock down both alleles: Transfect with a library of cDNA fragments designed to cover all mRNAs. Select for knockout phenotype (may require cleverness). Clone cells and recover RNAi to identify target gene.
A human near haploid cell strain. Use of it: Science, 326: 1231-1235 (2009) EMS = ethyl methanesulfonate: ethylates guanine UV (260nm): induces dimers between two adjacent pyrimidines on the same DNA strand
+ -+ -+ - + -
-+ + -
or
Heterozygote After homologous recombination(not sister chromatid exchange)
-- + -+
2 heterozygotes again
1 homozygote +/+
1 homozygote -/-
Homozygosis:Loss of heterozygosity (LOH)by mitotic recombination between homologous chromosomes (rare)
Paternal Chr. 4, say
Maternal Chr. 4
Recombinant chromatids
L RL R
LR RL
L L R R
Recessive phenotype is unmasked
= a mechanism of homozygosis of recessive tumor suppressor mutations in cancer
Mitosis
Mutagenesis (induced general mutations, not site directed)
Chemical and physical agents: MNNG point mutations (single base substitutions)EMS “ “Bleomycin small deletionsUV mostly point mutations but also large deletions Ionizing radiation (X-, gamma-rays) large deletions, rearrangements
Dominant vs. recessive mutations; Dom. are rare (subtle change in protein), but expression easily observed, Recessives are easier to get (whatever KO’s the protein function), but their expression is masked by the WT allele.
Categories of cell mutant selectionsExample
• Auxotrophs purine requiring
• Drug resistance Dominant ouabainR, alpha-amanitinR
Recessive 6TGr, BrdUr
• Antibodies vs. surface components MHC-
• Visual inspection G6PD-, Ig IP-
• FACS = fluorescence‑activated cell sorter DHFR-
• Brute force IgG-, electrophoretic shifts
• Temperature‑sensitive mutants 3H-leu resistant