Name: Protein Misfolding Cyclic Amplification of Prions (Video) | JoVE Journal
Uploaded: 2012-11-07T12:00:00.000+00:00
Duration: 10 min 12 s
Description: Learn Protein Misfolding Cyclic Amplification to study prion conversion and species barriers in vitro using hamster brain substrate. This method enables reliable detection and amplification of prion strains for neuroscience research.

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5 minutes

We would like to thank Dr. Vesper Fe Marie Ramos for critical reading of the manuscript. This work was supported by the National Center for Research Resources (P20 RR0115635-6, C06 RR17417-01 and G20RR024001) and the National Institute for Neurological Disorders and Stroke (2R01 NS052609).

1. Preparing the Equipment
<ol>
	<li>Use a Misonix 3000 or Misonix 4000 sonicator (Farmingdale, NY) connected to a Thermo Electron Neslab EX-7 water bath (Newington, NH) to keep a constant temperature of 37 &deg;C. Sonicate the samples in 200 &mu;l thin-walled PCR tube strips with domed caps obtained from Thermo Scientific (Waltham, MA).</li>
	<li>A brand new sonicator requires a "break-in" period of continuous operation9. A two-month break-in period consists of a 40-sec sonication burst and 10-minute incubation cycles with the amplitude level set to 10. One should observe erosion throughout the surface of the titanium microplate horn (Figure 1, panel B). The circulating water will look white turbid due to erosion of the titanium microplate horn. Replace this water daily.</li>
	<li>The amplification of PrPSc will vary throughout the microplate horn. The best PrPSc amplification efficiency can be obtained within a radius of 5 cm from the center of the microplate horn (See Figure 1, panel C).</li>
	<li>One round of PMCA consists 144 cycles. Each cycle consists of 5 sec sonication and 10 min incubation. This will roughly entail to 24 hr per round of PMCA.</li>
	<li>The power output of sonication, displayed on the sonicator's control panel, varies with the number of PCR tubes present in the sonicator's rack and the temperature of the circulating water from the water bath. Ensure that the water is equilibrated at 37 &deg;C, and do not fill more than 30% of the sonicator's tube rack (Figure 1, panel C). Spread the PCR tubes through the microplate horn. Have at least one row of empty space between PCR tube strips and allow no more than four tubes connected together per strip (Figure 1, panel C).</li>
	<li>The strength of sonication is critical for a successful prion strain amplification. While sonicating, adjust the amplitude to have a power output ranging between 155 ~ 170 watts. In our laboratory the power level is set to six and seven for the Misonix 3000 and Misonix 4000, respectively.</li>
	<li>Use distilled water in the water bath to avoid accumulating hard water stains. Replace this water weekly. During the incubation and sonication cycles, water condensation will occur inside the microplate horn lid. To avoid condensation, attach a small aquarium heat pad to the top of the sonicator's enclosure box as shown in Figure 1, panel A.</li>
</ol>
2. Preparing the Samples
<ol>
	<li>All procedures involving animals were approved by the Creighton University Institutional Animal Care and Use Committee and comply with the Guide for the Care and Use of Laboratory Animals. Before any tissue collection, the animal must be anesthetized and transcardially perfused using ice-cold phosphate buffered saline solution with 5 mM EDTA at pH 7.4 to remove blood from the brain and avoid the inhibition of PMCA reaction by blood7. Collect animal tissues rapidly using aseptic technique and have dedicated dissection tools for each prion strain. After dissection, place the tissue in a 1.5 ml microcentrifuge tube, flash freeze it on dry ice, and store it at -80 &deg;C until homogenization.</li>
	<li>To prepare the PMCA substrate (PrPC) solution, homogenize an uninfected hamster brain (UN BH) to a 10% weight/volume (w/v) solution with ice cold conversion buffer (see part 3) using a Tenbroeck tissue grinder. While homogenizing, be careful not to get air inside the grinder to avoid excessive foaming of the solution. Clarify the solution by centrifuging at 500 x g for 30 sec and aliquot the supernatant in 1.5 ml microcentrifuge tubes. Store at -80 &deg;C until further use.</li>
	<li>To prepare the PMCA seed (PrPSc) solution, homogenize an infectious material (brain, spleen, lymph nodes, other tissues or contaminated soil) to a 10% w/v solution with ice cold water and aliquot the solution in 0.6 ml microcentrifuge tubes. Store at -80 &deg;C until further use.</li>
</ol>
3. Preparing Conversion Buffer
<ol>
	<li>Weigh 0.5093 g of Triton X-100 in a 50 ml volumetric flask (result is a 1% final concentration of Triton X-100) (Sigma-Aldrich GmbH; Steinheim, Germany).</li>
	<li>Add one Complete Protease Inhibitor Tablet (Roche Diagnostics GmbH; Mannheim, Germany).</li>
	<li>Add 0.0931 g of EDTA (this will result in a 5 mM final concentration of EDTA) (Mallinckrodt Baker Inc.; Phillipsburg, NJ).</li>
	<li>Adjust the pH to 7.4 with 1N NaOH.</li>
	<li>Adjust volume to 50 ml with Dulbecco's Phosphate Buffered Saline (DPBS) (Mediatech Inc.; Manassas, VA).</li>
	<li>Filter the solution through a 0.45 &mu;m nitrocellulose membrane with a vacuum filtration apparatus. The conversion buffer can be stored at 4 &deg;C for no more than one month.</li>
</ol>
4. Amplification of Prion Strains Using PMCA
<ol>
	<li>Dilute the PMCA seed into the PMCA substrate at a ratio of 1:20 (i.e. mix 5 &mu;l of the PMCA seed and 95 &mu;l of the PMCA substrate) into a PCR tube. Remove and store a 15 &mu;l aliquot at -80 &deg;C for unsonicated control; this unsonicated control will be used to quantify the PrPSc amplification via proteinase K (PK) digestion followed by Western blot (WB) analysis. An additional unsonicated control could be generated by placing a second 15 &mu;l aliquot at 37 &deg;C for the duration of the PMCA reaction. A minimum of three repetitions is required for statistical purposes.</li>
	<li>Subject the remainder of the sample (70 &mu;l) to one round of PMCA as depicted on Figure 1, Panel A. Shake tubes every 5 hr to avoid water condensation on the domed cap of the PCR tube.</li>
	<li>The sample's tubes should be spun down and gently vortexed (being careful that the solution do not go into the domed cap) before opening after any procedure to ensure homogeneity and avoid potential contamination.</li>
	<li>For serial PMCA (sPMCA) of short incubation period strains (i.e. HY TME, HaCWD, or 263K), dilute the sonicated material at a ratio of 1:20 by transferring 5 &mu;l of the sonicated sample from round one into 95 &mu;l of fresh uninfected brain homogenate (Figure 2, Panel B).</li>
	<li>For sPMCA of long incubation period strains (i.e. DY TME, 139H, 22CH, 22AH, or ME7H), dilute the sonicated material at a ratio of 1:1 by transferring 50 &mu;l of the sonicated sample from round one into 50 &mu;l of fresh uninfected brain homogenate (Figure 2, Panel B).</li>
	<li>Remove two 15 &mu;l aliquots from the previously diluted sonicated mixture (from steps 4.4 or 4.5) and store one of them at -80 &deg;C and the other at 37 &deg;C (unsonicated control solutions for PMCA round two). Subject the remnants to a PMCA round two.</li>
	<li>This procedure can be repeated indefinitely. In our laboratory, we have performed up to fifteen PMCA rounds, with the strain properties remaining unchanged.</li>
	<li>PK digest the samples. For a typical WB, mix 5 &mu;l of PK 80 &mu;g/ml to 5 &mu;l of sample (this solution will have a final PK concentration of 40 &mu;g/ml) and incubate at 37 &deg;C for one hour. Add 10 &mu;l of gel loading buffer. Boil this solution for 10 min. Let the solution cool down and load 10 &mu;l into the gel.</li>
	<li>To calculate the success of amplification perform a WB analysis on the PK-digested samples to estimate the amount of amplified PrPSc by comparing them to either, the unsonicated controls20,21 Figure 2, Panel C or to known amounts of PK-undigested recombinant PrP.</li>
</ol>
5. Avoiding Cross-contamination
Critical steps have to be taken to minimize cross-contamination and the occurrence of false positives due to de novo formation of protease resistant products.
<ol>
	<li>Use a dedicated set of dissection tools (i.e. forceps, tweezers, scissors) for sample collection for each prion strain.</li>
	<li>Before homogenizing uninfected brains to be used as a PMCA substrate, wipe the pipettes with 1% bleach. Soak the sonicator's PCR tube rack, tissue homogenizers, and PCR tube storage racks with 1% bleach for 10 min and rinse thoroughly with distilled water.</li>
	<li>Cover the work area with a fresh Versi-Dry Lab Soaker every time a new sample is to be homogenized, a new PMCA experiment is to be prepared, or when aliquoting between sPMCA rounds.</li>
	<li>Use new gloves each time new samples are handled (especially when transferring aliquots between sPMCA rounds).</li>
	<li>Use short sonication bursts for each PMCA cycle. Cycles of 5 sec sonication combined with 10 min incubation is sufficient to amplify prions without generating de novo PrPSc 1,20,21,23,24 . As other laboratories have shown, increasing the sonication time, increasing the sonication amplitude, and extending the number of PMCA cycles increases the probability for de novo PrPSc formation2.</li>
</ol>

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<li>Barria, M. A., Mukherjee, A., Gonzalez-Romero, D., Morales, R., Soto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((De+novo+generation+of+infectious+prions+in+vitro+produces+a+new+disease+phenotype%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">De novo generation of infectious prions in vitro produces a new disease phenotype.</a> PLoS Pathog. 5, e1000421(2009).</li>
<li>Bessen, R. A., Marsh, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biochemical+and+physical+properties+of+the+prion+protein+from+two+strains+of+the+transmissible+mink+encephalopathy+agent%5BTitle%5D)+AND+%22J.+Virol%22%5BJournal%5D)">Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent.</a> J. Virol. 66, 2096-2101 (1992).</li>
<li>Bessen, R. A., Marsh, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+PrP+properties+suggest+the+molecular+basis+of+strain+variation+in+transmissible+mink+encephalopathy%5BTitle%5D)+AND+%22J.+Virol%22%5BJournal%5D)">Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy.</a> J. Virol. 68, 7859-7868 (1994).</li>
<li>Bessen, R. A., Marsh, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+two+biologically+distinct+strains+of+transmissible+mink+encephalopathy+in+hamsters%5BTitle%5D)+AND+%22J.+Gen.+Virol%22%5BJournal%5D)">Identification of two biologically distinct strains of transmissible mink encephalopathy in hamsters.</a> J. Gen. Virol. 73, 329-334 (1992).</li>
<li>Castilla, J., Gonzalez-Romero, D., Saa, P., Morales, R., De Castro, J., Soto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crossing+the+species+barrier+by+PrP%28Sc%29+replication+in+vitro+generates+unique+infectious+prions%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Crossing the species barrier by PrP(Sc) replication in vitro generates unique infectious prions.</a> Cell. 134, 757-768 (2008).</li>
<li>Castilla, J., Morales, R., Saa, P., Barria, M., Gambetti, P., Soto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell-free+propagation+of+prion+strains%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">Cell-free propagation of prion strains.</a> EMBO J. 27, 2557-2566 (2008).</li>
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<li>Dickinson, A. G., Fraser, H., Meikle, V. M., Outram, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Competition+between+different+scrapie+agents+in+mice%5BTitle%5D)+AND+%22Nat.+New+Biol%22%5BJournal%5D)">Competition between different scrapie agents in mice.</a> Nat. New Biol. 237, 244-245 (1972).</li>
<li>Gonzalez-Romero, D., Barria, M. A., Leon, P., Morales, R., Soto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+infectious+prions+in+urine%5BTitle%5D)+AND+%22FEBS+Lett%22%5BJournal%5D)">Detection of infectious prions in urine.</a> FEBS Lett. 582, 3161-3166 (2008).</li>
<li>Green, K. M., Castilla, J., Seward, T. S., Napier, D. L., Jewell, J. E., Soto, C., Telling, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Accelerated+high+fidelity+prion+amplification+within+and+across+prion+species+barriers%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">Accelerated high fidelity prion amplification within and across prion species barriers.</a> PLoS Pathog. 4, e1000139(2008).</li>
<li>Hadlow, W. J., Race, R. E., Kennedy, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temporal+distribution+of+transmissible+mink+encephalopathy+virus+in+mink+inoculated+subcutaneously%5BTitle%5D)+AND+%22J.+Virol%22%5BJournal%5D)">Temporal distribution of transmissible mink encephalopathy virus in mink inoculated subcutaneously.</a> J. Virol. 61, 3235-3240 (1987).</li>
<li>Kocisko, D. A., Come, J. H., Priola, S. A., Chesebro, B., Raymond, G. J., Lansbury, P. T., Caughey, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell-free+formation+of+protease-resistant+prion+protein%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Cell-free formation of protease-resistant prion protein.</a> Nature. 370, 471-474 (1994).</li>
<li>Kurt, T. D., Telling, G. C., Zabel, M. D., Hoover, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trans-species+amplification+of+PrP%28CWD%29+and+correlation+with+rigid+loop+170N%5BTitle%5D)+AND+%22Virology%22%5BJournal%5D)">Trans-species amplification of PrP(CWD) and correlation with rigid loop 170N.</a> Virology. 387, 3235-3240 (2009).</li>
<li>Maddison, B. C., Baker, C. A., Terry, L. A., Bellworthy, S. J., Thorne, L., Rees, H. C., Gough, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Environmental+sources+of+scrapie+prions%5BTitle%5D)+AND+%22J.+Virol%22%5BJournal%5D)">Environmental sources of scrapie prions.</a> J. Virol. 84, 11560-11562 (2010).</li>
<li>Nichols, T. A., Pulford, B., Wyckoff, A. C., Meyerett, C., Michel, B., Gertig, K., Hoover, E. A., Jewell, J. E., Telling, G. C., Zabel, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+protease-resistant+cervid+prion+protein+in+water+from+a+CWD-endemic+area%5BTitle%5D)+AND+%22Prion%22%5BJournal%5D)">Detection of protease-resistant cervid prion protein in water from a CWD-endemic area.</a> Prion. 3, 171-183 (2009).</li>
<li>Prusiner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+proteinaceous+infectious+particles+cause+scrapie%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Novel proteinaceous infectious particles cause scrapie.</a> Science. 216, 136-144 (1982).</li>
<li>Saborio, G. P., Permanne, B., Soto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensitive+detection+of+pathological+prion+protein+by+cyclic+amplification+of+protein+misfolding%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding.</a> Nature. 411, 810-813 (2001).</li>
<li>Saunders, S. E., Bartz, J. C., Vercauteren, K. C., Bartelt-Hunt, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+enzymatic+treatment+of+soil-bound+prions+effectively+inhibits+replication%5BTitle%5D)+AND+%22Appl.+Environ.+Microbiol%22%5BJournal%5D)">An enzymatic treatment of soil-bound prions effectively inhibits replication.</a> Appl. Environ. Microbiol. 77, 4313-4317 (2011).</li>
<li>Saunders, S. E., Shikiya, R. A., Langenfeld, K., Bartelt-Hunt, S. L., Bartz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Replication+efficiency+of+soil-bound+prions+varies+with+soil+type%5BTitle%5D)+AND+%22Journal+of+virology%22%5BJournal%5D)">Replication efficiency of soil-bound prions varies with soil type.</a> Journal of virology. , (2011).</li>
<li>Schutt, C. R., Bartz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prion+interference+with+multiple+prion+isolates%5BTitle%5D)+AND+%22Prion%22%5BJournal%5D)">Prion interference with multiple prion isolates.</a> Prion. 2, 61-63 (2008).</li>
<li>Shikiya, R. A., Ayers, J. I., Schutt, C. R., Kincaid, A. E., Bartz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Co-infecting+prion+strains+compete+for+a+limiting+cellular+resource%5BTitle%5D)+AND+%22Journal+of%22%5BJournal%5D)">Co-infecting prion strains compete for a limiting cellular resource.</a> Journal of. 84, 5706-5714 (2010).</li>
<li>Shikiya, R. A., Bartz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+generation+of+high+titer+prions%5BTitle%5D)+AND+%22Journal+of+virology%22%5BJournal%5D)">In vitro generation of high titer prions.</a> Journal of virology. , (2011).</li>
<li>Weber, P., Giese, A., Piening, N., Mitteregger, G., Thomzig, A., Beekes, M., Kretzschmar, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+genuine+prion+infectivity+by+serial+PMCA%5BTitle%5D)+AND+%22Veterinary+microbiology%22%5BJournal%5D)">Generation of genuine prion infectivity by serial PMCA.</a> Veterinary microbiology. 123, 346-357 (2007).</li>
</ol>

No conflicts of interest declared.

Challenges of amplifying infectious prion proteins are the long incubation periods and the expenses of in vivo experiments. The PMCA technique is a cost effective means to amplify infectious prion agents. Several laboratories have confirmed the ability of PMCA to accurately amplify prion strains in vitro 7, 9, 12, 14, 19,24 .
Prion diseases can be transmitted between species. Bessen and Marsh have effectively inoculated hamsters with transmissible mink encephalopat...

<table><tbody><tr><td>Misonix 3000</td><td>Misonix</td><td>S-3000</td><td></td></tr><tr><td>Misonix 4000</td><td>Misonix</td><td>S-4000</td><td></td></tr><tr><td>Tenbr&amp;#8211;ck Tissue Grinder</td><td>Kontes</td><td>885000-0007</td><td></td></tr><tr><td>Neslab EX-7 Water Bath</td><td>Thermo Electron</td><td>Neslab EX-7</td><td></td></tr><tr><td>0.2 ml PCR Tube Strips</td><td>Thermo Scientific</td><td>AB-0451</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>T9284-100ML</td><td></td></tr><tr><td>Complete Protease Inhibitor</td><td>Roche</td><td>11 697 498 001</td><td></td></tr><tr><td>EDTA</td><td>J.T. Baker</td><td>4040-00</td><td></td></tr><tr><td>DPBS</td><td>Mallinckrodt Baker Mediatech</td><td>21-031-CV</td><td></td></tr><tr><td>Versi-Dry Lab Soakers</td><td>Fisher Scientific</td><td>14 206 28</td><td></td></tr><tr><td>Repti Therm Heater</td><td>Zoo Med Laboratories, Inc.</td><td>RH-4</td><td></td></tr></tbody></table>

protein misfolding cyclic amplification of prions

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is used to amplify PrPSc in vitro7, 12, 14, 19, 24. A successful PrPSc amplification is shown by an increase in band intensity on Western blots of the PK-resistant prion protein (migrating between 19 and 30 kDa for hamster-derived prion strains) as shown in Figure 3. The increase in its band intensity after PMCA indicates amplification of the PK-resistant PrPSc material. Successful amplification of the ham...

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

The aim of this procedure is to successfully amplify hamster adapted PreOn strains in vitro. This is accomplished by first homogenizing uninfected hamster brain to be used as a substrate, and then preparing the infected tissue to be used as a seed. The second step is to mix the substrate and the seed in PCR tubes, and then perform protein misfolding, cyclic amplification or PMCA by sonication and incubation cycles.Next, the amplified material is diluted with fresh substrate and serially amplified in vitro, and then perform PMCA. Finally, the samples are digested with proteinase K and then analyzed by western blot. Ultimately, the increase in band intensity in the western blot can be used to determine the in vitro amplification of the prion strain.This method can help answer key questions to the PreOn field, such as what are the important co-factors involved in PreOn amplification? Generally, individuals new to this method will struggle with little but important details involving the technique, as well as to avoid sample cross-contamination. Before homogenizing wipe the pipettes with 1%bleach, saturate the sonicate PCR tube rack tissue homogenizers and PCR tube storage racks with bleach as well.After 10 minutes, rinse each piece of equipment thoroughly with distilled water and then cover the work area with a fresh Verly dry lab Soaker. Now use a 10 buric tissue grinder and freshly prepared ice cold conversion buffer to homogenize an uninfected hamster brain to a 10%weight per volume solution. For uses the PMCA substrate solution, then place the homogenate in 15 milliliter to conical tubes and clarify the homogenized solution by centrifugation at 500 times G for 30 seconds.Next, being careful to avoid the large pellet aliquot, the resulting snat into 1.5 milliliter micro centri tubes. To prepare the PMCA seed solution, homogenize harvested infectious material to a 10%weight per volume solution as just demonstrated, but with daros phosphate buffered saline or DPBS instead of conversion buffer, and then eloquent the solution into 0.6 milliliters of micro centrifuge tubes. Before beginning the amplification procedure, cover the work area with a fresh, firstly dry lab soaker at the PMCA substrate to a 200 microliter thin walled PCR tube strip and dilute the PMCA seed into it at a ratio of one to 20, place a total of four replicates in red labeled tubes.Place two replicates of a 15 microliter aliquot of each sample in blue labeled PCR tubes for use as a minus 80 degrees Celsius unsated control. Place an additional two replicates of a 15 microliter aliquot from each sample into PCR tubes to be stored at 37 degrees Celsius as an additional unsated control. Then place the minus 80 degrees Celsius unsated controls in a minus 80 degrees Celsius freezer and place the other unsated controls in a 37 degrees Celsius incubator for the duration of the PMCA reaction.Now begin the first PMCA amplification round by placing the PCR tube strip containing the remaining 70 microliters of each sample into a sonicate connected to a recirculating water bath set to a constant temperature of 37 degrees Celsius. A PCR tube containing PMCA substrate alone can be included as a mock control to check for cross-contamination nation fill no more than 30%of the sonata's tube rec by spreading the PCR tubes throughout the microplate horn. Keep at least one row of empty space between the PCR tube strips and allow no more than four tubes to be connected together per strip.The amplification of P-R-P-S-C varies throughout the microplate horn. The best P-R-P-S-C amplification efficiency is obtained within a radius of five centimeters from the center of the microplate horn. One round of PMCA consists of 144 cycles with five seconds of sonication and 10 minutes of incubation per cycle for about 24 hours per round of PMCA while fornicating adjust the amplitude to have a power output ranging between 155 to 170 watts.Place an aquarium heating pad on the sonicate lid to avoid water condensation, which could lead to sample cross-contamination. Shake the tubes every five hours to avoid water condensation on the domed caps of the PCR tubes after the round has been completed. Remove the tube strip from the ator and spin down the samples.Then gently vortex the tubes, keeping the solution from the cap to avoid contamination when the caps are opened to further minimize contamination when opening the tubes. Avoid touching the rim and place the cap on a clean Kim wipe. Then for serial PMCA of the PreOn strain transfer five microliters of the sonicated sample into 95 microliters of fresh PMCA substrate.Use new gloves for each sample. Next, remove two 15 microliter aliquots from each of the sonicated samples and store one aliquot minus 80 degrees Celsius and the other at 37 degrees Celsius. For the unsated control solutions for PMCA round two subject the remnants to a second PMCA round as just demonstrated.This step can be repeated as many times as desired. A PCR tube containing PMCA substrate of the uninfected brain alone can be included to check for cross contamination after syndicating the samples for the desired number of rounds, mix five microliters of 18 micrograms per milliliter of proteinase K with five microliters of the sonicated sample, the minus 80 degrees Celsius control and the 37 degrees Celsius. Control in separate PCR tubes and digest the sample at 37 degrees Celsius for one hour.Then add 10 microliters of gel loading buffer to the digested sample and boil the solution for 10 minutes. After it calls, load 10 microliters of the sample gel loading buffer mixture onto a Western block gel. To determine the amount of amplified P-R-P-S-C, compare the amount of proteinase k digested sample in the Western blot to either the unsated controls or to known amounts of proteinase.K undigested recombinant. P-R-P-C-P-M-C-A is used to amplify P-R-P-S-C in vitro successful amplification of the hamster adapted PreOn protein hamster CWD and drowsy TME can be seen in this western BLO analysis by comparing the band intensities before and after PMCA proteinase K digestion of P-R-P-S-C followed by Western BLO analysis shows an upper, middle and lower band corresponding to the dai mono and glycosylated prion protein respectively. Some PreOn strains can be differentiated by their electrophoretic mobility.For instance, there is a two kilodalton difference in migration between the hamster CWD and drowsy TME PreOn strains. This high fidelity amplification is observed by similar electrophoretic migration of the PMCA amplified PreOn hamster CWD strain compared to its corresponding hamster CWD seed as well as for the drowsy TME strain as compared to its drowsy TME seed. If no cross contamination or de novo, P-R-P-S-C formation occurs Western blot analysis, OFM control PMCA samples should remain clear after proteinase k digestion.While attempting to do this procedure, it is important to remember to clean every piece of equipment before using a new sample, thus avoiding cross contamination.

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Research

JoVE Journal

Immunology and Infection

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells.  Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Purification of Hsp104, a Protein Disaggregase

Incubate with a proteolytic enzyme to digest the normal prions, leaving the resistant aggregates intact.

Detection of Misfolded Prion Protein Aggregates in Mouse Brain Tissue Using Western Blotting

This video describes a degradation assay for misfolded proteins using cycloheximide treatment along with detergent fractionation. This method aids in studying the dynamics of misfolded proteins and uncovering the in-depth mechanisms of protein turnover.

Misfolding-Prone Protein Degradation Assay: A Technique to Monitor Misfolded Protein Degradation Using Cycloheximide Treatment and Detergent Fractionation

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Assays for the Degradation of Misfolded Proteins in Cells

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas.  We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Learn Protein Misfolding Cyclic Amplification to study prion conversion and species barriers in vitro using hamster brain substrate. This method enables reliable detection and amplification of prion strains for neuroscience research.

Protein Misfolding Cyclic Amplification of Prions

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