mapping functional prion-prion protein interaction sites using prion protein based peptide-arrays

Chapter 12

Mapping Functional Prion–Prion Protein Interaction SitesUsing Prion Protein Based Peptide-Arrays

Alan Rigter, Jan Priem, Drophatie Timmers-Parohi, Jan PM Langeveld,and Alex Bossers

Abstract

Protein–protein interactions are at the basis of most if not all biological processes in living cells. Therefore,adapting existing techniques or developing new techniques to study interactions between proteins are ofimportance in elucidating which amino acid sequences contribute to these interactions. Such new insightsmay in turn lead to improved understanding of the processes underlying disease and possibly provide thebasis for new therapeutic approaches. Here we describe the novel use of an ovine prion protein-basedpeptide-array normally used for determining prion-specific antibody epitopes, with the prospect that thiswould yield information on interaction sites between its PrP moiety and the ovine prion protein derivedlinear peptides. This adapted application of the peptide-array shows, by incubating the mature part of ovine(ARQ) PrPC fused to maltose binding protein (MBP), binding with between the PrP moiety and the ovineprion derived peptides occurs and indicates that several specific self-interactions between individual PrPmolecules can occur; hereby illustrating that this adapted application of a peptide-array is a viable methodto further specify which distinct amino acid sequences are involved in protein–protein interaction.

Key words: Protein–protein interaction, binding site, mapping, amino acid sequence, prion, peptide-array.

1. Introduction

Central in many biological processes is the interaction betweenproteins. This encompasses interaction with other as well as ‘‘self’’proteins, resulting in a multitude of effects. One example is signaltransduction, in which extracellular signals are conferred to theinside of a cell by protein–protein interactions of the signallingmolecules. Another is the long-term interaction between proteinsas part of a protein complex, in which one protein helps anotherfrom one to another cellular compartment (i.e. importins). Also

Marina Cretich, Marcella Chiari (eds.), Peptide Microarrays, Methods in Molecular Biology 570,DOI 10.1007/978-1-60327-394-7_12, ª Humana Press, a part of Springer Science+Business Media, LLC 2009

257

brief interactions between proteins occur, resulting in modifica-tion of the target protein (i.e. post-translational modification),which in itself can change protein–protein interactions. Thus,protein–protein interactions are of importance for most if not allbiological processes in living cells. Therefore, studying these inter-actions and elucidating the contributing amino acid sequencesinvolved in these interactions may lead to improved understandingof the processes underlying disease and may provide the basis fornew therapeutic approaches.

The common event in transmissible spongiform encephalopathies(TSEs) or prion diseases is the conversion of host-encoded proteasesensitive cellular prion protein (PrPC) into strain-dependent iso-forms of scrapie-associated protease resistant isoform (PrPSc) ofprion protein (PrP). Formation of PrPSc is a post-translationalprocess and involves refolding (conversion) of the host-encodedprion protein (PrPC) into partially protease resistant forms(PrPSc) (1).

These processes are determined by similarities as well asstrain-dependent variations in the PrP structure (2–11). Selectiveself-interaction between PrP molecules is the most probable basisfor initiation of these processes, potentially influenced by chaper-one molecules; however, the mechanisms behind these processesare far from understood. Here we describe the utilization of apeptide-array consisting of 15-mer overlapping peptides system-atically covering the whole mature part of the cellular prion protein(Fig. 12.1) in order to elucidate the interaction domains involvedin self-interaction of PrPC (12). To this end, an ovine PrP peptide-array consisting of 15-mer overlapping peptides was probed withrecombinant sheep PrPC fused to maltose binding protein(MBP–PrP).

1 256

455 well minicard

15-merpeptides

PrP-ORF

Fig. 12.1. Basic setup of the prion peptide-array. The mature PrP-ORF (excluding N- and C-terminal signal sequences) isdivided into 15-mer overlapping peptides, resulting in a grid where in each well the amino acid sequence has shifted byone amino acid.

258 Rigter et al.

2. Materials

2.1. MBP–PrP

Construction

and Verification

1. DifcoTM LB Broth (Miller)

2. DifcoTM LB Agar (Miller)

3. 100 mg/ml Ampicillin stock (Sigma, –20�C)

4. Amplitaq1 Gold DNA polymerase (Applied Biosystems)

5. GeneAmp1 PCR buffers (Applied Biosystems)

6. GeneAmp1 dNTP blend (Applied Biosystems)

7. Agarose (Merck)

8. TA cloning vector (Invitrogen, –20�C)

9. One Shot1 TOP10 Chemically Competent E. coli (Invitrogen,–20�C)

10. Wizard1 Plus Minipreps DNA Purification System (Promega)

11. Enzymes DraI and EcoRI (New England Biolabs, –20�C)

12. pMAL-c2X expression vector (New England Biolabs, –20�C)

13. Monoclonal PrP-specific antibody (9A2; in house, –20�C, seeNote 1)

14. Monoclonal MBP-specific antibody (New England Biolabs, –20�C, see Note 1)

15. Factor Xa (New England Biolabs)

16. 10% SDS stock (Merck)

17. NuPAGE gel-electrophoreses system (Invitrogen):a. XCell SureLock1 Mini-Cell

b. 12% NuPAGE1 Novex Bis-Tris Gel (4�C)

c. 20X NuPAGE1 MOPS SDS Running Buffer (4�C)

d. NuPAGE1 Antioxidant (4�C)

e. MagicMarkTM XP Western Protein Standard (4�C)

f. XCell IITM Blot Module CE Mark

g. NuPAGE1 Transfer Buffer (20X) (4�C)

h. Ureum sample buffer (4M ureum + 10% b-mercaptoetha-nol, –20�C)

18. HybondTM-C Extra (Amersham, optimized for protein transfer)

19. 0.05% PBS–Tween tablets (Calbiochem, RT)

20. SYPRO1 Orange protein gel stain (Invitrogen, 5000X con-centrate in DMSO, 4�C)

21. STORM 840/Typhoon (GE Healthcare, Life Sciences)

22. ECFTM substrate for Western blotting (GE Healthcare, –20�C, see Note 2)

Mapping Functional Prion–Prion Protein Interaction Sites 259

2.2. MBP–PrP

Expression and

Analysis

1. DifcoTM LB Broth (Miller, freshly made and autoclaved priorto use)

2. 100 mg/ml Ampicillin stock (Sigma, –20�C)

3. 2 g/l Glucose (Merck)

4. 0.1 M IPTG stock (Sigma, –20�C)

5. E. coli containing pMAL–MBP–PrP (glycerol-stock, –80�C)

6. Protease inhibitors (1000X stock) (–20�C, see Note 3):a. 25 mg/ml Pefabloc-Sc (AEBSF) in dH2O (500 mg,

Roche)

b. 700 mg/ml Pepstatine (1 mM) in MeOH (10 mg,Roche)

c. 500 mg/ml Aprotinine (0.15 mM) in dH2O (10 mg,Roche)

d. 500 mg/ml Leupeptine (1 mM) in dH2O (25 mg, Roche)

e. 0.5 M EDTA, pH 8.0 (Merck, RT)

7. Demineralized water (MiliQ)

8. Methanol, analysis grade (Merck)

9. Column-buffer (RT): 20 mM Tris-HCl pH 7.4, 200 mMNaCl, 1 mM EDTA pH 8.0, 10 mM b-mercaptoethanol(added fresh prior to use).

10. Cuphorn sonicator (Branson)

11. 10% Triton X-100 stock (BDH, RT)

12. Amylose slurry (New England Biolabs, supplied pre-swollenin 20% ethanol, 4�C)

13. 5 ml Disposable columns (Qiagen)

14. 1 M Maltose stock (Merck, 4�C)

15. 0.5 ml siliconized screw-cap tubes (BioPlastics)

16. NuPAGE gel-electrophoreses system (Invitrogen)

17. HybondTM-C Extra (Amersham, optimized for proteintransfer)

18. 0.05% PBS–Tween tablets (Calbiochem, RT)

19. SYPRO1 Orange protein gel stain (Invitrogen, 5000X con-centrate in DMSO, 4�C)

20. 10% non-immune goat serum (Zymed laboratories inc.)


22. STORM 840/Typhoon Trio (GE Healthcare, Life Sciences)

23. ECFTM substrate for Western blotting (GE Healthcare, –20�C, see Note 2)

260 Rigter et al.

2.3. Peptide-Array

Analysis

Materials needed for synthesis and coupling of 15-mer pep-tides to form the ovine prion protein peptide-array are to acertain extent described in (13–16); however, the exact meth-odological details are the intellectual property of Pepscan Sys-tems BV, who perform these experiments on a contractualbasis. Measurement of colouration of the substrate and sub-sequent translation to the optical density requires a specializeddigital camera and analysis software setup as described bySlootstra et al. (15). The whole procedure, except for subse-quent analysis of the final density values, was performed byPepscan Systems BV.

1. SQ-buffer (4�C): 5% (v/v) horse serum, 5% (v/v) henalbumin, 1% (v/v) Tween 80 in PBS


3. Rabbit anti-mouse-IgG antibody coupled horse radish per-oxidase antibody (Dako, 4�C)

4. 0.05% PBS–Tween (Calbiochem, RT)

5. ABTS peroxidase substrate (Sigma-Aldrich)

6. Hydrogen peroxide (Merck, H2O2)

7. CCD camera and image processing system:a. Sony CCD Video Camera XC-77RR

b. Nikon micronikkor 55 mm f/2.8 lens

c. Camera adaptor (Sony Camera adaptor DC-77RR)

d. Image processing softwarepackage TIM, v. 3.36 (DifaMeasuring Systems, Breda, The Netherlands).

3. Methods

3.1. MBP–PrP

Construction and

Verification

Polymerase chain reaction (PCR) was performed conforming tostandard conditions. Cloning of the PCR-product into theTA-cloning vector and sub-cloning into pMAL-c2X wereperformed conforming to the protocols supplied by themanufacturers.

1. In order to obtain the PrP gene suitable for cloning into thepMAL protein fusion and purification system, the mature partof the ovine PrP (ARQ) open reading frame (ORF) was PCRamplified using platinum Taq polymerase and PrP specificprimers ShBo-F-DraI (50-GGTGGTTTTAAAAAGCGAC-CAAAACCTGG-30) and Sh-R-STOP (50-GGTGGTCTA


TGCCCCCCTTTGGTAATAAGCC-30) using standard50 ml PCR reaction containing: 100 ng DNA template, 5 mlPCR buffer (10X), 0.5 ml dNTPs (50 mM), 1.0 ml of eachprimer (10 mM), sterile water to a total volume of 49 ml and 1unit of Taq polymerase.

2. In order to check the integrity and purity of PCR product,5.0 ml of the PCR reaction was run on a 1% agarose gel. ThePCR produced a single band of appropriate size; therefore,the PCR product was directly cloned into the TA-vector (seeNote 4).

3. The resulting PrP (AA25-233), without its N- and C-terminal signal sequences, was cloned into a general TA-cloning vector using the TA Cloning1 Kit, by adding1.0 ml of fresh PCR-product to 50 ng of the TA-vector,1.0 ml of 10X ligation buffer, 1.0 ml of T4 DNA Ligase(4 Weiss units) and make up to a total volume of 10.0 mlwith sterile water.

4. After ligation of the PCR-product in the TA-vector, thecomplete ligation reaction was transformated in the commer-cial TOP10 chemically competent E. coli and thetransformated cells spread on LB-agar plates containing100 mg/ml ampicillin.

5. Single colonies were picked and grown overnight at 37�Cwhile shaking in 2 ml LB-broth containing 100 mg/mlampicillin.

6. Plasmid DNA was isolated using the Wizard1 Plus MiniprepsDNA Purification System for further use.

7. Before sub-cloning the mature PrP ORF in the pMAL-c2Xexpression plasmid, the PrP ORF was sequenced to excludePCR artefacts.

8. The PrP fragment was subsequently sub-cloned using DraIand EcoRI into the pMAL-c2X expression vector, resulting inthe maltose binding protein (MBP) fusion to the N-terminusof PrP (MBP–PrP).

In order to assess whether the complete mature part of PrPC

was expressed correctly and whether the fusion protein had acces-sible folding, MBP–PrP was expressed and analysed as described inSection 3.2. MBP–PrP was digested with factor Xa as described inthe manual of the pMAL protein fusion and purifications system.Expression of maltose binding protein N-terminally fused to PrP(MBP–PrP) revealed a mainly soluble recombinant MBP–PrP ofapproximately 70 kDa (Fig. 12.2, lanes 1 and 2) and the nakedPrP protein could be obtained by digestion with protease Fac-tor Xa, indicating accessible folding (Fig. 12.2, lanes 3–6).After 24 h approximately 45% of MBP–PrP was digested byfactor Xa; however, when aided by addition of 0.01% SDS

262 Rigter et al.

factor Xa completely digested MBP–PrP within 24 h (data notshown). Furthermore, MBP–PrP is readily detected in Westernblot using both a PrP-specific antibody (9A2, Fig. 12.2B) anda MBP-specific antibody (Fig. 12.2C). Several monoclonal andpolyclonal antibodies with recognition sites dispersed through-out the PrP protein detected MBP–PrP in Western blot, indi-cating complete expression of the mature part of sheep PrPC

(data not shown). MBP expressed without additional fusionprotein (PrP), which frequently served as negative control inthis study, was also of homogeneous quality (Fig. 12.2, lane 7)and of expected size (MBP-b-gal a fragment, 50.8 kDa) whichis somewhat larger (as expected) than MBP cleaved from thefusion protein after factor Xa digestion (42.5 kDa).

- MBP

- MBP-PrP

- MBPC

- PrPB

- MBP-PrP

80 -

kDa

- PrP

60 -

50 -

40 -

30 -

20 -

100 -120 -

220 -

A

- intact factor Xa

- heavy chainof factor Xa

- MBP-β-gal α frag.

1 2 3 4 5 6 7

- MBP-β-gal α frag.

1 2 3 4 5 6 1 2 3 4 5 6 7

Fig. 12.2. Analysis of MBP–PrP and MBP expression and MBP–PrP digestion by Factor Xa. Lane 1 contains untreatedMBP–PrP, whereas lane 2 contains a mock digestion of MBP–PrP. MBP–PrP was digested with 1% w/w factor Xa andduring digestion samples were taken at 2, 4, 7 and 24 h (lanes 3,4, 5 and 6, respectively). All samples were run on SDS-PAGE and the gel was stained with Sypro Orange (total protein stain, panel A) before Western blotting and subsequentimmunodetection using either a PrP-specific monoclonal antibody (9A2, panel B) or MBP-specific monoclonal antibody(a-MBP, panel C). Expression of MBP, expressed from the pMAL-c2X vector with no insert (MBP-b-gal a fragment), wasanalysed by Western blot using either 9A2 or a-MBP (lane 7, panels A and C, respectively). Reproduction of Fig. 1,published in BMC Biochemistry by Rigter et al. (12).


3.2. MBP–PrP

Expression and

Analysis

Expression and purification by affinity chromatography wasperformed as described in the manual of the pMAL protein fusionand purifications system (method I) with the followingadaptations:

1. The litre of rich broth (LB-broth containing 2 g/l glucoseand 50 mg/ml ampicillin) was inoculated with 50 ml over-night culture.

2. To improve binding of MBP–PrP by preventing formation ofinterchain disulphide bonds upon lysis (as suggested in theprotocol), 10 mM b-mercaptoethanol was added to the col-umn buffer.

3. Before sonication of the cell suspension, an inhibitor cocktail(pefabloc sc, pepstatine, aprotinine, leupeptine and EDTA)was added and the cell suspension was sonicated eight timesfor 15 s, resting the cell-extract on ice for 45 s betweensonication rounds.

4. To ensure optimal lyses of the cells 1% (v/v) Triton X-100 wasadded to the sonicated cell suspension and incubated for20 min at 4�C, while gently rocking.

5. After lyses the dilution of the crude extract with columnbuffer was not necessary.

6. A column was prepared using 2 ml of 20% amylase slurry andthe MBP–PrP fusion protein was eluted after binding withcolumn buffer containing 10 mM maltose in 10 fractions of250 ml.

Quantity and quality of the eluted MBP–PrP was determinedby SDS-PAGE as follows.

1. From each collected fraction a 10 ml sample was taken and runon a 12% NuPAGE1 Novex Bis-Tris Gel together with abovine serum albumin (BSA) concentration curve samples(2.0, 1.0, 0.5 and 0.25 mg, see Note 5).

2. After separation by electrophoresis, the gel was taken out ofits casing, trimmed and washed in 1X NuPAGE1 transferbuffer for 10 min to remove excess unbound SDS from thegel (see Note 6).

3. The gel was then transferred to fresh 1X NuPAGE1 transferbuffer containing 1:5000 diluted Sypro1 Orange and incu-bated for 1 h (see Note 7).

4. The gel was then rinsed in 1X NuPAGE1 transfer buffer andscanned using the STORM 840 using standard settings (seeNote 8).

5. After scanning, the gel was blotted for 1 h at constant voltageof 30 V onto nitrocellulose (HybondTM-C Extra) using theXCell IITM Blot Module CE Mark and NuPAGE1 transferbuffer (see Note 9).

264 Rigter et al.

6. The resulting blot was transferred to a 50 ml falcon tubecontaining 40 ml blocking buffer (5% milk protein in PBS)and incubated for 1 h at room temperature while gentlyshaking. Alternatively the blot can be incubated overnightat 4�C.

7. After blocking the blot was rinsed once in 40 ml of 0.05%PBS–Tween.

8. The blot was then incubated in 5 ml PBS–Tween containing1% non-immune goat serum (NGS) and either 1:1500diluted MBP-specific antibody or 1:1000 diluted PrP-specificantibody 9A2 as primary antibody, for 1 h at room tempera-ture while gently shaking.

9. After incubating with the primary antibody, the blot wasrinsed once in 40 ml of PBS–Tween (0.05%).

10. After rinsing the blot was washed two times in 25 ml 0.05%PBS–Tween for 10 min while gently shaking.

11. The blot was then incubated in 5 ml 0.05% PBS–Tweencontaining 1% NGS and 1:2000 diluted goat-anti-mouse-IgG coupled with alkaline phosphatase as the secondaryantibody, for 1 h at room temperature while gentlyshaking.

12. After incubating with the secondary antibody, the blot wasrinsed once in 40 ml of 0.05% PBS–Tween.

13. After rinsing the blot was washed two times in 25 ml 0.05%PBS–Tween for 10 min while gently shaking.

14. The 0.05% PBS–Tween of the final wash is discarded and50 ml of fresh 0.05% PBS–Tween was added for transportand storage at 4�C (see Note 10).

15. The surface area of the blot was determined and 24 ml/cm2

ECF was pipetted directly onto the glass plate of the STORM840.

16. The blot was taken out of the 0.05% PBS–Tween in which itwas stored, and gently dried before placing the blot onto theECF, making sure the ECF spreads underneath the wholeblot (see Note 11).

17. The blot was immediately scanned using the STORM 840scanning software and standard settings (see Note 12) forECF and the scan was repeated after 5, 10, 15 and ifnecessary 20 min (see Note 13) of the start of the initialscan.

18. Using the ImageQuant software supplied by themanufacturer, the band density was determined for furtheranalysis.


3.3. Peptide-Array

Analysis

Synthesis of complete sets of overlapping 15-mer peptides(Fig. 12.1) were carried out on grafted plastic surfaces, coveringthe ovine PrP amino acid sequence of mature PrPC (residues 25–234 of ovine and 25–242 of bovine PrP) (17). The plastic surfaceconsisted of 455, 3 ml wells on a credit-card size plastic (minicard)carrier. In each well the peptides were coupled to the graftedplastic surface in two ways: either by direct step-by-step couplingof amino acids or by coupling complete 15-mer peptides at theirC-terminus (13–16) and subsequent ELISA analyses were per-formed. The established procedures for peptide-array analysis areused to determine antibody recognition domains (Fig. 12.3A);however, when using the prion protein peptide-array to determineinteraction domains of PrP, direct detection with a-PrP antibodiescan possibly recognize peptides that did not bind PrP and thusfrustrate the results. However, by incubating MBP–PrP as theantigen that binds to the peptide, detection of the fusion proteinis possible using a-MBP antibody, followed by rabbit a-mouse-HRP (Fig. 12.3B), preventing frustration of the results due tocross-reaction between the primary antibody and the ovine prionprotein derived peptides. The procedure described below is notextensive; however, points 2–4 entail the necessary adaptation ofthe standard peptide-array analysis protocol, which allows forelucidation of the amino acid sequences involved in protein–pro-tein interaction.

1. Support-coupled peptides were pre-coated with SQ-bufferfor 1 h at 37�C to block non-specific absorption of antibodies.

α−PrP RαM-POpeptide

PrP(26 kDa)

M-α-MBP RαM-POPrP-MBPpeptide

A

α−PrP RαM-POpeptide

B

PrP(26 kDa) MBP (44kDa)MBP (44kDa)

M-α-MBP RαM-POPrP-MBPpeptide

Fig. 12.3. ELISA-based detection principle of peptide-array analysis. (A) Standarddetection setup for a peptide-array in order to determine antibody epitope; (B) Alternativesetup used to determine peptide–protein interaction in this study.

266 Rigter et al.

2. MBP–PrP was diluted in SQ-buffer (standard buffer used byPepscan B.V.) to a concentration resulting in a sufficientrelative density value, allowing differentiation between peaks(Fig. 12.4, column graph), without affecting the backgroundand preventing the relative density value reaching its plateau.In the case of MBP–PrP a concentration of 7.5–10 mg/ml wasoptimal (see Note 14).

3. MBP–PrP was incubated overnight at 4�C as an antigen onthe peptide-array minicard.

4. The peptide-array minicard was washed three times in0.05% PBS–Tween and all buffer was removed from thewells.

5. The peptide-array minicard was incubated with the primaryMBP-specific antibody diluted 1:2000 in SQ-buffer for 1 h at25�C.

24 36 48 60 72 84 96 108

120

132

144

156

168

180

192

204

216

228

PK CHO CHO

H1 H2 H3S1 S2GPI-signal

peptideS S

signal peptideI II III IV V

peptide number/amino acid position

relative den

sity value

3

7

11

15

19

0

1

2

4

−2

−1

rela

tive

hyd

rop

hili

city

3

101 113 137 149 161 173 185 197 22129 41 53 65 8917 77 125 209

Fig. 12.4. Overview of PrPC secondary structures and antibody epitopes versus peptide-array binding pattern and Kyte–Doolittle hydrophilicity plot. Schematic representation of PrPC showing signal sequences, b-sheets (S1, S2), a-helices(H1, H2, H3), disulphide bridge site (S–S) and glycosylation sites (CHO). The sequence of PrP is lined up with both theKyte–Doolittle hydrophilicity plot (line graph; negative is hydrophobic and positive is hydrophilic) and the relative bindingpattern found (column graph) with the ovine prion peptide-array. Adaptation of Fig. 5, published in BMC Biochemistry byRigter et al. (12).


6. The peptide-array minicard was washed three times in0.05% PBS–Tween and the buffer was removed from allthe wells.

7. The peptide-array minicard was incubated with thesecondary rabbit anti-mouse-IgG antibody coupled horseradish peroxidase antibody diluted 1:1000 in SQ-buffer for1 h at 25�C.

8. The peptide-array minicard was washed three times inPBS–Tween (0.05%) and the buffer was removed from allthe wells.

9. After washing the peroxidase substrate ABTS and 2 ml/ml3% H2O2 was added and incubated for 1 h at roomtemperature.

10. The optical density of the signals in the separate wells wemeasured using a CCD camera and an image processingsystem as described by Slootstra et al.(15).

11. The measured density values were further analysed as follows:The background was determined by calculating the mean

value of 20 peptides with low density values of which at least 5peptides were in consecutive order. The relative density value(r.d.v.) was calculated by dividing the optical density value(o.d.v.) by the background and binding was considered rele-vant when at least three consecutive peptides showed bindingvalues of at least three times the background.

Interaction between the individual PrP sequences (peptides) andMBP–PrP was sufficient for immunodetection, resulting in areproducible binding pattern (Fig. 12.4, column graph). Thisbinding pattern, expressed in relative density values was character-ized by two distinct high binding areas (peptides 35–102 and 134–102, respectively) as well as some lower binding areas. Analysis ofthe correlating peptide sequences revealed that these areas usuallywere characterized by consensus sequences which suggested theexistence of distinct interaction domains for the mature part ofPrPC (for detailed analysis see (12)). To further assess the extent ofthe specificity of the binding pattern found, MBP–PrP was alsotested against a bovine PrP peptide-array. This yielded a similarbinding pattern comparable to the results with ovine PrP peptide-array but with some slight differences, which can be correlated todifferences in amino acid differences between the ovine and bovineprion protein (12) (data not shown).

To find a correlation with structural properties, the relativebinding pattern of MBP PrP on the peptide-array was com-pared to the Kyte–Doolittle hydrophilicity plot of mature PrPC

(Fig. 12.4, line graph) revealing a high correlation betweenhydrophilic (exposed) regions of PrPC and the observedbinding pattern regions.

268 Rigter et al.

4. Notes

1. Storage of the antibody stocks is done best at –20�C. Stocksare best divided in aliquots before storage. When an aliquot istaken into use it is best to store this aliquot at 4�C to preventantibody degradation by repeated thawing and freezing untilfinished.

2. ECF is best stored as aliquots of 1 ml (about two blots) tominimize decline of the substrate as a result of repeatedthawing and freezing.

3. For ease of use it is best to aliquot the protease inhibitors(except EDTA) and store at –20�C, for prolonged sto-rage. The aliquot in use can be stored at 4�C untilfinished.

4. When the PCR does not produce a single band it is pertinentto run the whole PCR reaction on an agarose gel and excisethe band that correlates to the specific PCR product wanted.Extract the DNA from the gel size using a commerciallyavailable kit. However, the amount of PCR product recoveredis usually low and if possible it is advised to optimize the PCRreaction in order to get a single band. Then the proceduredescribed can be followed, with greater chance of success.

5. After staining with Sypro Orange and visualization using theSTORM 840, the ImageQuant software supplied by themanufacturer can be used to determine the (relative) densityof the BSA bands, these densities (‘‘volumes’’) can be extra-polated against the concentration of BSA loaded in the lane,resulting in a (linear) concentration curve. From this curvethe concentration of the protein present in the fractions canbe estimated using the determined band volumes for thesefractions.

6. Wash at least for 10 min, but no more than 30 min. SyproOrange binds to protein-bound SDS. Washing helpseliminate background colouring; however, prolongedwashing diminishes the protein staining.

7. Sypro Orange is light sensitive; therefore, incubating in aclosed light-impermeable container is advised for optimalresults.

8. Sypro Orange has an excitation max of 300 and 470 nm, andan emission max of 570 nm; use Blue fluorescence mode(automatically coupled to emission filter) on STORM 840.

9. Correct stacking of the gel, nitrocellulose and blot-pads isdescribed in the information provided by the manufacturer.


10. The blot can be stored at 4�C in PBS–Tween for several weekswithout loss of signal. However, storage may cause the signalto come up later compared to direct measuring using ECFand the STORM 840.

11. When placing the blot onto the ECF, make sure no bubblesare trapped underneath the blot. It is easier to add extra ECFwhen necessary afterwards, when to much ECF is present thiswill flow out from underneath the blot and will create smear-ing during scanning.

12. ECF has an excitation max of 440 nm and an emission max of560 nm; use the Blue fluorescence mode (automaticallycoupled to emission filter) on STORM 840.

13. Scanning at different time-intervals allows for choosing thescan in which good signal is present, but in which the highestsignal has not yet reached its plateau. This ensures that thesubsequent density measurements of the bands can be com-pared in further analysis.

14. During the experiments we discovered that soluble MBP–PrPprotein interacts with the peptides on the peptide-array.Aggregation of MBP–PrP resulted in disappearing of signal;therefore, slight differences in optimal MBP–PrP concentra-tion are likely due to differences in solubility between differ-ent batches of MBP–PrP used.

Acknowledgements

This work was supported by a grant 903-51-177 from the DutchOrganization for Scientific Research (NWO), by a grant from theDutch Ministry of Agriculture, Nature Management and Fisheries(LNV) and by EU NeuroPrion project FOOD-CT-2004-506579. We kindly thank Saskia Ruiter for her work on theMBP–PrP expression construct.

References

1. DeArmond, S. J., and Prusiner, S. B. (2003)Perspectives on prion biology, prion diseasepathogenesis, and pharmacologicapproaches to treatment. Clin Lab Med,23, 1–41.

2. Atarashi, R., Sim, V. L., Nishida, N.,Caughey, B., and Katamine, S. (2006)Prion strain-dependent differences in con-version of mutant prion proteins in cell cul-ture. J Virol, 80, 7854–62.

3. Bossers, A. (1999) PhD-Thesis: ‘‘Prion dis-eases: Susceptibility and transmissibility. Invivo and in vitro studies with sheep scrapie’’.June 10th1999, University of Utrecht, TheNetherlands. ISBN 90–393–2108–6,http://library.wur.nl/WebQuery/wur-pubs/lang/309746.

4. Bossers, A., Belt, P., Raymond, G. J.,Caughey, B., de Vries, R., and Smits, M. A.(1997) Scrapie susceptibility-linked

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polymorphisms modulate the in vitro con-version of sheep prion protein to protease-resistant forms. Proc Natl Acad Sci USA,94, 4931–6.

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mapping functional prion-prion protein interaction sites using prion protein based peptide-arrays

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