ReadyPrep™ Reduction-Alkylation Kit

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ReadyPrep Reduction-Alkylation Kit

Overview

Reduction and Alkylation

Disulfide bond removal using the ReadyPrep™ reduction-alkylation kit. Protein samples were reduced using either 50 mM DTT in rehydration/sample buffer (before) or the reduction-alkylation kit (after). Both samples were applied by cup loading onto 11 cm ReadyStrip™ pH 7–10 IPG strips and focused for the first dimension, then run on Criterion™ 8–16% Tris-HCl precast gels for the second dimension.

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The ReadyPrep reduction-alkylation kit reduces and irreversibly blocks the formation of inter- and intramolecular disulfide bonds prior to isoelectric focusing (IEF). Disulfide bond formation is particularly problematic for basic proteins because of the increased rate of their formation in an alkaline environment. Moreover, many reducing agents become negatively charged during IEF and migrate off the IPG strip, thus allowing disulfide bonds to re-form (Görg 2000, Hoving 2002).

The ReadyPrep reduction-alkylation kit:

  • Eliminates the potential for disulfide bond formation during IEF
  • Reduces disulfide bonds with tributylphosphine (TBP) and alkylates the sulfhydryl groups with iodoacetamide (Herbert et al. 2001)
  • Provides reagents for treatment of 100 samples (300 µl each, containing up to 10 mg/ml of protein)
  • Performs treatment directly in the rehydration/sample buffer, in less than 2 hours, and with very little hands-on time
  • Is compatible with other ReadyPrep sample preparation kits and buffers

References
Görg A et al. (2000). The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21, 1,037–1,053.
Herbert B et al. (2001). Reduction and alkylation of proteins in preparation of two-dimensional map analysis: Why, when and how? Electrophoresis 22, 2,046–2,057.
Hoving S et al. (2002). Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients. Proteomics 2, 127.

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ReadyPrep™ Reduction-Alkylation Kit, 100 preps

1632090
100 preps, includes 3 x 0.6 ml 200 mM TBP, 3 empty glass vials for TBP storage, 5 x 56 mg iodoacetamide, 2 ml alkylation buffer, instruction manual; for 100 x 300 µl samples containing up to 10 mg/ml protein

0 1632090 /templatedata/internet/product_SKU/data/LSR/Protein-Sample-Preparation/Protein-Sample-Cleanup/163-2090_0921120744002.xml 1632090 09/21/09 12:07 PM N en 09/21/19 12:07 PM true true internet/product_SKU AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA LSR 2018-02-01 00:53:58 1632090-readyprep-reduction-alkylation-kit-100-preps 1632090 ReadyPrep Reduction-Alkylation Kit ReadyPrep&trade; Reduction-Alkylation Kit, 100 preps 100 preps, includes 3 x 0.6 ml 200 mM TBP, 3 empty glass vials for TBP storage, 5 x 56 mg iodoacetamide, 2 ml alkylation buffer, instruction manual; for 100 x 300 µl samples containing up to 10 mg/ml protein 100 preps, general purpose protein preparation kit for reducing gel streaking and improving resolution of basic proteins 100 preps, includes 3 x 0.6 ml 200 mM TBP, 3 empty glass vials for TBP storage, 5 x 56 mg iodoacetamide, 2 ml alkylation buffer, instruction manual; for 100 x 300 µl samples containing up to 10 mg/ml protein /webroot/web/images/lsr/products/protein_sample_preparation/sku_thumb/global/163-2090_tn.jpg /webroot/web/images/lsr/products/protein_sample_preparation/sku_view/global/163-2090_view.jpg /webroot/web/images/lsr/products/protein_sample_preparation/sku_feature/global/163-2090_feature.jpg ReadyPrep&trade; Reduction-Alkylation Kit, 100 preps preview RepEx 163-2090 100 preps, general purpose protein preparation kit for reducing gel streaking and improving resolution of basic proteins 09/21/09 12:07 PM 09/21/19 12:07 PM AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA en LSR /LSR/Protein-Sample-Preparation/Protein-Sample-Cleanup N 0 <p>Choose the ReadyPrep Reduction-Alkylation Kit to reduce disulfide bonds and block their formation in isoelectric focusing (IEF) samples. The kit contains tributylphosphine (TBP) to reduce both intramolecular and intermolecular disulfide bonds, and iodoacetamide to then alkylate the thiols.</p> <ul> <li>Improve IEF resolution and reproducibility</li> <li>Eliminate disulfide bond formation during IEF</li> <li>Obtain a consistent spot pattern, and minimize streaking and aberrant spot migration</li> <li>Use directly in rehydration/sample buffer; complete in less than 2 hours, with very little hands-on time</li> <li>Compatible with other ReadyPrep sample preparation kits and buffers</li> </ul> <p>The ReadyPrep Reduction-Alkylation Kit is a simple and effective method to improve the results of 2-D electrophoresis. Many reducing agents become negatively charged during isoelectric focusing and migrate off the IPG strip.&nbsp; Furthermore, dithiothreitol (DTT), the most commonly used reducing agent, is not effective under acidic conditions. TBP has been shown to be more effective reducing agent than DTT for 2-D gel electrophoresis. Additionally, alkylation improves the resolution of basic proteins, which have an increased rate of disulfide bond formation in an alkaline environment (G&ouml;rg et al. 2000, Hoving et al. 2002).</p> <p>The combination of reduction with TBP and the subsequent alkylation of the thiols with iodoacetamide is effective in reducing intramolecular and intermolecular disulfide bonds and preventing the reformation of disulfide bonds and disulfide shuffling, resulting in higher resolution, less spot ambiguity, and increased reproducibility.</p> <h2 class="descHeader">Kit Components</h2> <p>For treatment of 100 samples, each 300 &micro;l containing up to 10 mg/ml protein</p> <ul> <li>TBP (200 mM), 0.6 ml, 3 ampoules</li> <li>Empty glass vials for TBP storage after opening the ampoules, 3</li> <li>Iodoacetamide, 56 mg, 5 amber vials</li> <li>Alkylation buffer, 2 ml</li> <li>Instruction manual </li> </ul> <h2 class="descHeader">Related Products</h2> <ul> <li>ReadyPrep Kits for <a href="/en-us/category/fractionation-cellular-location?">Fractionation by Cellular Location</a></li> <li>ReadyPrep Kits for <a href="/en-us/category/fractionation-solubility">Fractionation by Solubility</a></li> <li>Aurum&trade; Ion Exchange Mini Kits for <a href="/en-us/category/fractionation-charge">Fractionation by Charge</a></li> <li>Rotofor<sup>&reg;</sup> Liquid-Phase IEF Cells for <a href="/en-us/category/fractionation-pi">Fractionation by pI</a></li> <li>Model 491 Prep Cell and Mini Prep Cell for <a href="/en-us/category/fractionation-solubility">Fractionation by Solubility </a></li> <li><a href="/en-us/product/readyprep-2-d-starter-kit">ReadyPrep&trade; 2-D Starter Kit</a></li> <li><a href="/en-us/product/protean-i12-ief-system">PROTEAN<sup>&reg;</sup> i12&trade; IEF System</a></li> <li><a href="/en-us/product/readystrip-ipg-strips">ReadyStrip&trade; IPG Strips</a></li> <li><a href="/en-us/product/powerpac-hv-high-voltage-power-supply">PowerPac&trade; HV High-Voltage Power Supply</a></li> </ul> <h2 class="descHeader">Related Categories</h2> <ul> <li><a href="/en-us/category/protein-gels">Protein Gels</a></li> <li><a href="/en-us/category/electrophoresis-chambers">Electrophoresis Chambers</a></li> <li><a href="/en-us/category/2-d-electrophoresis?ID=6916a228-0f95-4b26-a37a-5e4afe76add7-buffers-reagents">2-D Electrophoresis Buffers and Reagents</a></li> <li><a href="/en-us/product/ampholytes">Ampholytes</a></li> <li><a href="/en-us/category/tube-gel-ief-systems">Tube Gel IEF Systems</a></li> <li><a href="/en-us/category/imaging">Gel Imaging Systems</a></li> <li><a href="/en-us/product/2-d-electrophoresis-workflow">2-D Electrophoresis Workflow</a></li> </ul> <h2 class="descHeader">Supporting Documentation</h2> <ul> <li>ReadyPrep Reduction-Alkylation Kit <a href="/webroot/web/pdf/lsr/literature/MS4110063A.pdf" target="_blank">Instruction Manual</a></li> <li>Sample Preparation: Tools for Protein Sample Extraction, Cleanup, Fractionation, and Depletion <a href="/webroot/web/pdf/lsr/literature/Bulletin_3096.pdf" target="_blank">Brochure</a></li> <li>ReadyStrip IPG Strip <a href="/webroot/web/pdf/lsr/literature/4006166H.pdf" target="_blank">Instruction Manual</a></li> <li>PROTEAN i12 IEF System <a href="/webroot/web/pdf/lsr/literature/Bulletin_6097A.pdf" target="_blank">Brochure</a></li> <li>Host Cell Protein (HCP) Workflow <a href="/webroot/web/pdf/lsr/literature/Bulletin_6493.pdf" target="_blank">Quick Guide</a></li> </ul> <h2 class="descHeader">References</h2> <p>G&ouml;rg A et al. (2000). The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21, 1037&ndash;1053. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/10786879">10786879 </a><br />Hoving S et al. (2002). Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients. Proteomics 2, 127. PMID: <a href="http://www.ncbi.nlm.nih.gov/pubmed/11840558">11840558</a></p> /webroot/web/html/lsr/products/SKUs/autopopulated 1632090.html /webroot/web/html/lsr/products/SKUs/autopopulated/1632090.html false false false /prd/en/CA/adirect/biorad?cmd=ajaxAddToCart&AddToCartProductID=1632090&CartQuote=QT&quantity= true true false $311.00 false null /prd/en/CA/adirect/biorad?cmd=ajaxAddToCart&AddToCartProductID=1632090&AddToDefaultWishList=true&quantity= true false true true false true true true /prd/en/CA/adirect/biorad?cmd=ajaxAddToCart&AddToCartProductID=1632090&quantity= 1632090
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ReadyPrep™ Proteomics Grade Water

1632091
500 ml, ultrapure water for use with ReadyPrep fractionation and general purpose cleanup kits

3 1632091 /templatedata/internet/product_SKU/data/LSR/Electrophoresis-and-Blotting/Experion-Automated-Electrophor/163-2091_0921120319815.xml 1632091 09/21/09 12:03 PM N en 09/21/19 12:03 PM true true internet/product_SKU AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA LSR 2017-11-14 07:08:10 1632091-readyprep-proteomics-grade-water 1632091 ReadyPrep Proteomics Grade Water ReadyPrep™ Proteomics Grade Water 500 ml, ultrapure water for use with ReadyPrep fractionation and general purpose cleanup kits 500 ml, ultrapure water for use with ReadyPrep fractionation and general purpose cleanup kits 500 ml, ultrapure water for use with ReadyPrep fractionation and general purpose cleanup kits /webroot/web/images/lsr/products/protein_sample_preparation/sku_thumb/global/163-2091_tn.jpg /webroot/web/images/lsr/products/protein_sample_preparation/sku_view/global/163-2091_view.jpg /webroot/web/images/lsr/products/protein_sample_preparation/sku_feature/global/163-2091_feature.jpg preview RepEx 163-2091 500 ml, ultrapure water for use with ReadyPrep fractionation and general purpose cleanup kits 1632091, readyprep, proteomics, grade, water, ultrapure, fractionation, general, purpose, cleanup 09/21/09 12:03 PM 09/21/19 12:03 PM AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA en LSR /LSR/Electrophoresis-and-Blotting/Experion-Automated-Electrophor N 0 false false false /prd/en/CA/adirect/biorad?cmd=ajaxAddToCart&AddToCartProductID=1632091&CartQuote=QT&quantity= false true false $56.00 false null /prd/en/CA/adirect/biorad?cmd=ajaxAddToCart&AddToCartProductID=1632091&AddToDefaultWishList=true&quantity= true false true true false true true true /prd/en/CA/adirect/biorad?cmd=ajaxAddToCart&AddToCartProductID=1632091&quantity= 1632091
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Iodoacetamide

1632109
30 g, alkylating agent for use in protein sample preparation applications

4 1632109 /templatedata/internet/product_SKU/data/LSR/Electrophoresis-and-Blotting/Protein-Electrophoresis-and-Bl/163-2109_0921120349272.xml 1632109 09/21/09 12:03 PM N en 09/21/19 12:03 PM true true internet/product_SKU AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA LSR 2017-11-14 07:08:25 1632109-iodoacetamide 1632109 Iodoacetamide Iodoacetamide 30 g, alkylating agent for use in protein sample preparation applications 30 g, alkylating agent for use in protein sample preparation applications 30 g, alkylating agent for use in protein sample preparation applications /webroot/web/images/lsr/products/electrophoresis/sku_thumb/global/163-2109_tn.jpg /webroot/web/images/lsr/products/electrophoresis/sku_view/global/163-2109_view.jpg /webroot/web/images/lsr/products/electrophoresis/sku_feature/global/163-2109_feature.jpg RepEx 163-2109 30 g, alkylating agent for use in protein sample preparation applications 09/21/09 12:03 PM 09/21/19 12:03 PM AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA en LSR /LSR/Electrophoresis-and-Blotting/Protein-Electrophoresis-and-Bl N 0 false false false /prd/en/CA/adirect/biorad?cmd=ajaxAddToCart&AddToCartProductID=1632109&CartQuote=QT&quantity= true true false $208.00 false null /prd/en/CA/adirect/biorad?cmd=ajaxAddToCart&AddToCartProductID=1632109&AddToDefaultWishList=true&quantity= true false true true false true true true /prd/en/CA/adirect/biorad?cmd=ajaxAddToCart&AddToCartProductID=1632109&quantity= 1632109
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Tributylphosphine (TBP)

1632101
0.6 ml, 200 mM, reducing agent for use in protein sample preparation applications

5 1632101 /templatedata/internet/product_SKU/data/LSR/Electrophoresis-and-Blotting/Protein-Electrophoresis-and-Bl/163-2101_0921120349250.xml 1632101 09/21/09 12:03 PM N en 09/21/19 12:03 PM true true internet/product_SKU AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA LSR 2017-11-14 07:08:25 1632101-tributylphosphine-tbp 1632101 Tributylphosphine (TBP) Tributylphosphine (TBP) 0.6 ml, 200 mM, reducing agent for use in protein sample preparation applications 0.6 ml, 200 mM, reducing agent for use in protein sample preparation applications 0.6 ml, 200 mM, reducing agent for use in protein sample preparation applications /webroot/web/images/lsr/products/electrophoresis/sku_thumb/global/163-2101_tn.jpg /webroot/web/images/lsr/products/electrophoresis/sku_view/global/163-2101_view.jpg /webroot/web/images/lsr/products/electrophoresis/sku_feature/global/163-2101_feature.jpg RepEx 163-2101 0.6 ml, 200 mM, reducing agent for use in protein sample preparation applications 09/21/09 12:03 PM 09/21/19 12:03 PM AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA en LSR /LSR/Electrophoresis-and-Blotting/Protein-Electrophoresis-and-Bl N 0 false false false /prd/en/CA/adirect/biorad?cmd=ajaxAddToCart&AddToCartProductID=1632101&CartQuote=QT&quantity= false true false $62.00 false null /prd/en/CA/adirect/biorad?cmd=ajaxAddToCart&AddToCartProductID=1632101&AddToDefaultWishList=true&quantity= true false true true false true true true /prd/en/CA/adirect/biorad?cmd=ajaxAddToCart&AddToCartProductID=1632101&quantity= 1632101
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4110063 Instruction Manual, ReadyPrep™ Reduction-Alkylation Kit, Rev A Click to download

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