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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

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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

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A Biacore result

Ligand added

Ligand removed

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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:

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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

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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

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a phosphatase

FK506 recruits FKBP to bind to calcineurin and inhibit its action as a specific phosphatase

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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

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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

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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