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±NE.coli°ö¾i¦bLB¤¤, 37¢J, 300rpm, ®¶Àú°ö¾i¹L©],
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6.
¸Ë§´1²ÕNucleoSpin®M²Õ,
±N²GÅé§l¤J, Â÷¤ß10,000rpm, 2min, Ë¥h²GÅé, «¸Ë§´NucleoSpin®M²Õ.
7.
¥[¤J500£gl BW, Â÷¤ß10,000rpm, 1min, Ë¥h²GÅé, «¸Ë§´NucleoSpin®M²Õ.
8.
¥[¤J600£gl B5, Â÷¤ß10,000rpm, 1min, Ë¥h²GÅé, «¸Ë§´NucleoSpin
®M²Õ.
9.
Â÷¤ß13,000rpm,
3min, Ë¥h²GÅé, «¸Ë§´NucleoSpin®M²Õ©ó1¤ä·sªº1.5mlÂ÷¤ßºÞ¤¤.
10.¥[¤J200£gl¹w¼ö¦Ü70¢JªºBE buffer, ¸m«Ç·Å2min, Â÷¤ß13,000rpm,
1min, ©Ò±o§YDNA.
Y©Ò±oDNA¤§A260/280<1.6, ¥i¥[¤Jµ¥¶q(1volume)B3¤Îµ¥¶q99¢Malcohol,
vortex 10sec, ¦A±N²GÅé§l¤JNucleoSpin ®M²Õ¤¤, Â÷¤ß13,000rpm, 1min,
Ë¥h²GÅé, ¦A¥[¤J500£gl BW²G, Â÷¤ß10,000rpm,
1min, Ë¥h²GÅé, ¦A¥[¤J600£gl
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¥[¤J200£gl¹w¼ö¦Ü70¢JªºBE
buffer, ¸m«Ç·Å2min, Â÷¤ß13,000rpm, 1min, ©Ò±o§YDNA.
.¤Q.¨Ï¥ÎCoulter
Counter Z1¤èªk
¨Ï¥Î«e¥ý±NDiluent ²~¸Ë¤JIsotonII¦Ü¤Ö¥b²~, ±NWaste²~˪Å.
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¨Ï´ú¸ÕºÞ¤Î¹qªMªw¦bIsoton¤º.
1.
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«Ý§¹¦¨«á;
2.
«öset-up,
½T»{aperture¬°100£gm, Kd=60, select Units¬°£gm, lower size=2.0£gm, volume=0.5ml, ¦A«öset-up,
¾÷¾¹·|½Õ¾ã³]©w¤§°Ñ¼Æ, ¦A«öset-up, ¬Ý³]©wȹï§_, ¹ï, «h¦A«öset-up,
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±NCoulter Cleanz²G¸Ë¤J´ú¸ÕªM, «öPrime, ¾÷¾¹·|²M¬~ºÞ¤l8¦¸, ¦A´«dH2O¸Ë¤J´ú¸ÕªM,
«öPrime, ¾÷¾¹·|¦A²M¬~ºÞ¤l8¦¸, µM«áÃö¾÷.
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®Æ¡G
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²GºA´á
Extraction
buffer
[°t¤è]100mM
Tris.Cl, pH8 100mM
EDTA
250mM
NaCl
100£gg/ml proteinase K
10¢M(W/V)
N-lauroyl sracosine (SarKosyl)
100¢M¤Î70¢Methanol
TE
buffer
7.5M
ammonium acetate
¤è ªk ¤@¡G
1.
¨ú¥®¹à´Óª«²Õ´10~30g,
¥HdH2O²M¬~, ´½°®.
2.
¥H²GºA´á±N´Óª«²Õ´§Ná,
¿i¦¨¯».
3.
±N¯»¸Ë¤JÂ÷¤ßºÞ,
¥[¤Jextraction buffer (¨C§J´Óª«²Õ´¥[10ml extraction buffer),
¥R¤À²V¦X. ¦A¥[¤J¾A¶q¤§10¢MSarKosyl¨Ï³Ì²×¿@«×¦¨¬°1¢M.
4.
¸m55¢J,
1hr.
5.
Â÷¤ß10min,
6,000rpm, 4¢J, «O¯d¤W²M, ¦Ü¥t¤@Â÷¤ßºÞ¤¤.
6.
¥[¤J¤W²M²G0.6¿ªºisopropanol½w½w²V¦X.
DNAÀ³§YªR¥X, §_«h¸m-20¢J,
30min.
7.
Â÷¤ß8,000rpm,
4¢J, 15min, Ë¥h¤W²M. ¦A±NDNA·»©ó¾A¶qTE¤¤.
8.
±µ¤U¨Ó¥i¥Î¯Â¤ÆDNAªº¤èªk¯Â¤ÆDNA
(§Y25¡G24¡G1¤§Phenol/Chloroform/isoamyl alcohol)µ¥¶q²V¦X, Â÷¤ß2,800rpm,
10min, ¤W²M²G¤¤¬°DNA.
9.
±N¤W²M²GÂà¦Ü¥t¤@Â÷¤ßºÞ¤¤,
¥[¤J1/2¿Åé¿n¶q¤§7.5M ammonium acetate, ¥H¤Î¤W²M²G¶q2¿¤§100¢Methanol,
ªR¥XDNA. ¦A¥H2,800, 2min, Â÷¤ß, ¨I¾ýDNA, Ë¥h¤W²M.
10.¥Î70¢Methanol¬~DNA,
¥H¥h°£ÆQ¤À§Yphenol, Ë¥h°sºë, ±NDNA·»©ó¾A¶qTE¤¤.
11.±NDNA«O¦s¦b-20¢J.
¥Î¦¹ªk©Ò±o¤§DNA·|§t¦³²É¸¢Åé¤Î¸ºñÅ餤ªºDNA,
n·Q±o¨ì¯Â´Óª«DNA, n¥Î¶W°ª³tÂ÷¤ß¾÷¤ÀÂ÷.
¥H¦¹ªk©Ò±o¤§DNA¶q,
¤j¬ù¬O¨C§J´Óª«10¦Ü40£gg DNA.
1.
¨ú25g´Óª«¥®¸¬~²b, ¸m¬ã²Ú¤¤,
¥[¤J25mlÂfÂc»Ä¯Ç²G(°t¤è¡G¨C¤½¤ÉdH2O¤¤¥[8.18g Na
citrate°ª·Å°ªÀ£®ø¬r), ¬ã¿i¦¨ªdª¬.
2.
¥[¤J150ml§¡½è¤Æ²G(Homogenization
Solution) (°t¤è¡G¨C¤½¤ÉdH2O¤¤¥[Na lauryl sulfate 100.0g, Na
citrate 2.94g, NaCl 8.18g, °ª·Å°ªÀ£®ø¬r), ¦AÄ~Äò¬ã¿i30min,
±N¦¹ªdª¬²G¥HÂù¼h¯½¥¬¹LÂo, ¨ÃÀ½¥X¯½¥¬¤Wªº²GÅé.
3.
±N²GÅé¾A¶q§¡¤À¸mÂ÷¤ßºÞ,
¤À§O¥[¤J2¿Åé¿nªº99¢M°sºë, Â÷¤ß200xg, 5min, (4¢J), Ë¥h¤W²M,
¦¹¨I¾ý¤¤§t²ÓM®Ö, ¥[¤Jì²GÅé¿n1.5¿¶qªºNaCl²G
(°t¤è¡G¨C¤½¤ÉdH2O¤¤¥[NaCl 81.8, °ª·Å°ªÀ£®ø¬r),
¥R¤À²V¦X, Â÷¤ß10,000xg, 25min, 20¢J,
±N¤W²M²GˤJ¤@²M¼äµLµß¤p¿NªM, «O¯d.
4.
¦A¥[¤J15ml NaCl²G¦ÜÂ÷¤ßºÞ¤¤,
¥R¤À²V¦X, ¦AÂ÷¤ß10,000xg, 25min,
20¢J.
5.
±N¤W²M²GˤJ«e¤w¦³¤W²M²GˤJªº¦P¤@²M¼äµLµß¤p¿NªM¤¤,
½w½w¥[¤Jµ¥Åé¿nªº99¢M°sºë, ¨Ã¥H¬Á´ÎÅÍ©Õ, ¦¹®ÉDNA·|¶¦A¬Á´Î¤W,
Ä~ÄòÅÍ©Õ, ¦ÜDNA¤£¦A¶¦b¬Á´Î¤W.
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Prep AquaPure Genomeic DNA KitµÑ¨ú²ÓµßDNA
1.
¨úE.coli
overnight culture 500£gl, ¸m¤J1.5mlÂ÷¤ßºÞ,
¸m¦B¤W, Â÷¤ß10,000rpm 15sec, §l°£¤W²M²G, ¦A¥[¤J300£gl
genomic lysis buffer, ¨Ï²Óµß¥R¥÷·»¸Ñ, ¸m80¢J¤ô¯D©Îhotblock¤W, 5min.
2.
±N¤WzÂ÷¤ßºÞ¸m«Ç·Å5min,
¥[¤J1.5£gl RNase solution,¥R¥÷²V¦X,¸m37¢J, 45min,
¦A±N¤WzÂ÷¤ßºÞ¸m«Ç·Å5min, ¦A¥[¤J100£gl protein precipitation
solution, vortex 30sec, Â÷¤ß11,000rpm, 3min.
3.
«O¯d¤W²M²G©ó¥t¤@Ó1.5mlÂ÷¤ßºÞ¤¤,
¥[¤J300£gl 100¢Misopropanol, ¥R¥÷²V¦X, Â÷¤ß13,000rpm, 2min,
Ë¥h¤W²M, ¥H300£gl 70¢M°sºë½w¬~¨H¾ý¤§DNA,
Â÷¤ß13,000rpm, 1min, Ë¥h¤W²M, Ãw°®.
4.
¥[¤J50~100£gl DNA hydration buffer, ¸m65¢J¤ô¯D©Îhotblock¤W,
5~30min. ¨ÏDNA¥R¤À·»¸Ñ. «O¦s©ó-20¢J.
.¤Q¥|.´Óª«²Õ´°ö¾i»P¥Íª«§Þ³N
±N±ý°µ²Õ´°ö¾iªº´Óª«²M¬~°®²b, ¦A®û¤J70¢M°sºë20¬í,¦A®û¤J§t0.05¢MTween
20©ÎTriton-100ªº0.25¢Mº}¥Õ¤ô(NaOCl), ©Î3¢MH2O2,
©Î1¢MAgNO3¤¤20min,(¥i¸m©ó¶Wµªi¼Ñ¤¤³B²z).
¦A¥HµLµß»]ÃH¤ô®û¬~3¦¸, ¨C¦¸5min.
¡¯¥H¬õù¸²¬°¨Ò¡G
±N¬õù¸²¥~ªí¬~²M«á,
¥HµLµß¸Ñå¤M, ¤Á¥X¤@Á¡¶ê¶ô, ¦A¥H¤Wz¨BÆJ®ø¬r«á, ¦A¤Á¥X1¤p¶ô§Î¦¨¼h(cambium)³¡¥÷,
¦¹¤p¶ô¥i¦A¤À¤Á¬°¦h¶ô, ¤À¸mµLµß¥B§t¡¶Ë²Õ´»¤µo²G(Callus-induction
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pH¡×5.5[¥Î0.1M NaOH½Õ]), Yn©TºA«h¥[¤J8.0g agar, °ª·Å°ªÀ£®ø¬r)ªºµLµß¦³»\¸ÕºÞ¤¤,
°ö¾i²G¤£¶W¹L¤p¶ôªí±, ¸m«Ç·Å, ¤é¥ú¿O¤U, ¬ù4¶g,
¥i¨£Â¡¶Ë²Õ´.
¥H¥É¦ÌF¬°¨Ò¡G
¥ý±N¥É¦Ì²É¥H«e×ä¤è¦¡®ø¬r,
¦A¸mµLµß°ö¾i¥×¤¤, ¸m«Ç·Å·t³B, ¥HµLµß»]ÃH¤ô®û1¤é, ²Ä2¤é,
¥HµLµß¸Ñå¤M, ±NF¥H¥~³¡¥÷¤Á°£, ±NFÂಾ¦Ü§tF°ö¾i°ò(Embryo
Culture Medium)¥½L¤¤ (°t¤è¡G1x MS medium 1¤½¤É, pH¡×5.5[¥Î0.1M NaOH½Õ]), Yn©TºA«h¥[¤J8.0g agar,
°ª·Å°ªÀ£®ø¬r, «Ý·Å«×°¦Ü50¢J¥ª¥kË¥½L). ¥½L©P³ò¥Hparafilm«Ê¦n,
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¥H§ß®á¸¬°¨Ò¡G
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¥H«e×ä¤è¦¡®ø¬r, ¦A¨ú¤¤¶¡¦³¸¯ß³¡¥÷, ¤Á¦¨1cm2¤p¶ô,
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´Óª«ì¥Í½èÅé¬O¤£¨ã²ÓM¾Àªº´Óª«²ÓM.
¥H§ùÃYªá¬°¨Ò¡G
¨ú§ùÃYªá¨Ì«e×äªk®ø¬r«á,
¤Á¦¨¬ù1mm¼e±øª¬, ¸mµLµß°ö¾i¥×¤¤§t10ml²ÓM¾Àµõ¸Ñ²G(Cell Wall
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¦A¥H¹LÂoªk®ø¬r), ¸m«Ç·Å·L¥ú¤U1¤é. ²Ä2¤é,¥i±N°ö¾i¥×¸m¸ÑåÅã·LÃè¤U,¥HµLµß§lºÞ±Nì¥Í½èÅé§l¥X¥t¦æ°ö¾i©óMS²G¤¤,¦A¨ú¤Ö³\¥H0.3¢Mtrypan
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buffer, ¥ý¥R¥÷²V¦X«á, ¦A¸m¥»O³¼±×¾¹¤W½w½w²V¦X5min, Â÷¤ß3,000rpm,
5min, §l°£¬õ¦â¤W²M²G.
2.
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4.
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¦b°µ¥Õ¦å²y¬V¦âÅé®É¤w¤jP¤¶²Ð¹L¡G
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±N¥i°ª·Å°ªÀ£®ø¬rªºMEM¥[¤JÀ³¦³Á`¶q90¢MªºdH2O,
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(Y«Yºû«ù²G, ¤û¦å²M¶q¥i°u´î).
2.
Animal cell lines¥i¦V·s¦Ë¹¬ì©Ò±ÄÁÊ.
3.
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µM«á§l°£2/3, «O¯d1/3, ¦A¥[¤J4.3ml°ö¾i²G, §Y¥i.
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reaction mixture, ±N¤w²G¤Æ¤§DNase1 ¨ú10£gl, ¥[90£gl DNase 1 reaction
buffer, ±N¦¹²V¦X²G§l¤J3¤§¤ººÞ¤¤, ¸m«Ç·Å15min.
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ª`·N:
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«h¥[¤J1,000£gl RM1 binding buffer (¥i¾A¥Î©ó100~1,000£gg¤§RNA¶q), vortex¨ÏRNA¥R¤À²V¦X.
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7.
¦A¥[¤J500£gl washing buffer RM3©óNucleoSpin microfilter¤¤,
¨Ã¤p¤ß¤£§Ë¯}NucleoSpin microfilter¦Ó±NOlig dT²É¤l¥R¤À²V¦X. Â÷¤ß15sec,
5,000rpm, ¦AÂ÷¤ß2min, 10,000rpm. Ë¥h²GÅé(¦A¦¸¥h°£rRNA).
8.
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13,000rpm, ¥H°®ÀêNucleoSpin microfilter. ±NNucleoSpin microfilterºÞ¨ú¥X¸m©ó¥t1RNase-freeÂ÷¤ßºÞ¤¤.
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dT²É¤l²G, ¥[¤J20£gl¤w¥[¼ö¦Ü68¢JªºRNase-free
water, §l¤W§l¤U, ¥R¤À²V¦X, ¸m68¢J, 7min, Â÷¤ß1min, 13,000rpm.
©Ò±o²GÅé§YmRNA. ¥i¦A§l¤Jì³Ìªì¥[¤J¨C10£glªºOlig dT²É¤l²G,
¥[¤J10£gl¤w¥[¼ö¦Ü68¢JªºRNase-free
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¥i¤À§O«O¦s¦b-70¢J.
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1.
·Ç³Æ2.5mMªºdNTPmix»P5x
Reverse Transcriptase (RT) buffer(§t250mM Tris-Cl, pH8.3, 375mM KCl, 15mM MgCl2),
RT 200£gg/£gl, 0.1M DTT(³q±`ÀH¶RRT¨Ãªþ),
10x Taq polymerase buffer.
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·Ç³ÆmRNA, (³Ì¦n1£gg/£gl), 10£gl, ¥[¤J1£gl(0.1£gg/£gl)¤urandom primer(6-mer), ¸m70¢J,
2min.
3.
¥[¤J5x
RT buffer 5£gl, 0.1M DTT
3.5£gl, dNTPmix 5£gl, RT 1£gl, ¸m37¢J1hr.
4.
¸m70¢J,
5min, ¨ÏRT¯}Ãa.
5.
¨ú2.5£gl¥X¨Ó¥Î¸m¤J¥t1PCR¥Î0.5mlÂ÷¤ßºÞ, ¥[¤J8£gl dNTPmix, 10£gl Taq polymerase buffer,
¥[¤J°w¹ï¦¹°ò¦]³]pªº2Óprimers¦U1£gl, ¥[¤J1£gl Taq polymerase,
¦A¥[¤JdH2O 77£gl, ¦A¥[50£gl
mineral oil, °õ¦æPCR, 94¢J,
5min, 94¢J, 1min, 55¢J, 1min, 72¢J, 2min, 30cycles, §Y¦¨.
©Ò±o¦¨«~¥i¥H¹qªa¯Â¤Æ.
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®Æ¡G
¹qªa½w½Ä²G(¥i¥ÎTAE©ÎTBE),
°t¤è¦p¤U¡G
TAE-Àx¦s²G¡G50X,
¨C¤½¤É¤¤§t¡G242g Tris. Base
57.1ml
glacial acetic acid
37.2g Na2EDTA. 2H2O
pH8.5
¨Ï¥Î²G¡G0.04M
Tris. Acetate
0.002M EDTA
TBE-Àx¦s²G¡G10X,
¨C¤½¤É¤¤§t¡G108g Tris. Base
55g boric acid
40ml
0.5M EDTA
pH8.0
¨Ï¥Î²G¡G0.089M
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0.089M boric acid
Ethidium
bromide solution
[°t¤è]1000XÀx¦s²G(0.5mg/ml)
50mg
ethidium bromide
100ml
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«O¦s¦b4¢J,
¤£³z¥ú²~¤¤.
¨Ï¥Î²G¡G0.5£gg/ml
±NÀx¦s²G¥H1¡G1000¿µ}ÄÀ§@¥Î
Agarose
10X
loading buffer
[°t¤è]
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0.1¢M
Sodium dodecyl sulfate
0.25¢M
Bromphenol blue
0.25¢M
Xylene Cyanol
¨ä¤¤Xylene Cyanol ¦b¹qªa®É, ¤ñBromphenol
blue¶]±oºC50¢M¥ª¥k.
DNA¤À¤l¶qmarker(¥i¨Ì¹qªa¤§DNA¤À¤l¤j¤p¿ï¾Ü¾A·í¤§marker)
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loading buffer«á, ¥HmicropipetÂà¤J½¦¤Õ¤¤.
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buffer¤§ÂŦⲾ¦Ü¬Û·í¦ì¸m«á, §Y¥i±N¹q¬yÃö±¼, Àˬd¹qªaµ²ªG,
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modifier¤Î4.5¿Åé¿nªºNaI.
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¤£§tethidium bromide.
¦]¬°TBE¹ïDNAÖߪþ¦bEZ-GLASSMILK¤W¦³¼vÅT,
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¨C¹j¤@¤ÀÄÁ¨ú¥X²V¦X¤@¤U, ¦A©ñ¤J, ¬ù5¤ÀÄÁªa½¦¥i§¹¥þ¿Ä¤Æ.
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µM«á¶i¦æ¤U¦C¨BÆJ].
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2.
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10sec.
4.
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Y¬O¥Î¦P¤@Ó酶¦P®É¤Á³Î³\¦hDNA¼Ð¥»,
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¥i¦V¼t°Ó±ÄÁʲV¦X¼Ð·Ç²G©Î¦Û¤v±N¤wª¾¤À¤l¶qªº³J¥Õ½è²V¦X¨Ï¥Î.
¨Ò¦p¡GBSA
¤À¤l¶q66,000
ªþµù¡G
1.
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·U·|Æp¤Jmatrix(¶ñ®Æ)ªº¤Õ¤¤, ©µ¿ð¬y¤U¨Óªº³t«×,
¥ç¥i±Ä°ª®Ä²G¬Û¼hªR»ö(HPLC, high-performance liquid chromatography)³B²z,
¦ý¨Ï¥ÎHPLC®É, void volume¬OBed volumeªº1/2¦Ó«D1/3.
2.
Y¤£·»©ó¤ôªº³J¥Õ½è,
¥i¸Õ·»©ó6M guanidine.Cl¤Î8M urea²G¤¤.
3.
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¤£¨Ï¥Î´Á¶¡, ¨¾¤î²Óµß´F¥Í, ¥i¦bbuffer¤¤¥[¤J0.02¢MªºSodium
azide, ¦ý¦b¨Ï¥Î«eÀ³¥Îbuffer±N¦¹§tsodium azideªº²GÅé¨R°£.
4.
¨Ï¥Î¤§¼Ë¥»¶q¤£©y¦h¹Lvolumeªº5¢M.
5.
¬y³t¤£©y¤p¹L2cm/hr©Î¤j¹L10cm/hr.
6.
¶ñ¥Rª«¦b90¢J,
¬ù5¤p®É¥i¿±µÈ¦Ü¥i¥Îµ{«×.
7.
¬Wª¬¼hªR°£¥H¤À¤l¤j¤p¼hªR¥~,
¤W¦³¥HÂ÷¤l¥æ´«¼hªR, §K¬Ì¿Ë©M¼hªRµ¥¤è¦¡, ¦³¾÷·|¦A¦æ¤¶²Ð.
.¤T¤Q¤.¥HHPLC¯Â¤Æ³J¥Õ½è
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HPLC¾A©ó·L¶q³J¥Õ½èªº¤ÀÂ÷,
¥B¥i¦bµu®É¶¡¤º¹F¦¨. °f¬Ûªk³õ·|¨Ï³J¥Õ½è¤À¤l¥¢¥h¥Íª«¬¡©Ê,
Â÷¤l¥æ´«»P¹½¤ô©Êªk§¡µL¦¹¯ÊÂI,
¤À¤l¤j¤pªk©Ò¤Àªº¤À¤l¤j¤p½d³ò·|¤ñ´¶³q¥Îªº¬Wª¬¼hªR¤p¤@¥b,
¥B¥i¹ï«Ü¤p¶qªº¼Ë¥»¶i¦æ¤ÀÂ÷.
¦¹³B¶È¤¶²Ð¥H¤À¤l¤j¤p¨Ó¤ÀÂ÷³J¥Õ½èªº¤èªk.
¦¹ªk¨Ì³J¥Õ½è¤À¤l¤j¤p, ¤j¤À¤l¥ý¤À¥X, ¤p¤À¤l«á¤À¥X.
§÷
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degassed,
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size-exclusion(SE)
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SE
protein standards
0.75¡Ñ30cm
SE column
¤è
ªk¡G
1.
±N«O¦s²G(storage
solvent) (¦¹³B¨Ï¥Îmethanol)¥Ñcolumn¤¤¬~°£(¥Îdegassed HPLC-grade H2O),
¥Hflow rate 1ml/min.
(¦b¨Ï¥Î§¹¤§«á, ¥H¤ô¬~¥hbuffer salts, flow rate
1ml/min 30min, ¦A¥Hmethanol¬~column 30min, 1ml/min)
2.
¦b«Ç·Å¤U¨ÏSE
column¥¿Å©ó1ml/min, H2O¤¤.
3.
¸Õ´ú¤@¦¸,
¥H100£gl SE buffer¨Ó´ú,
±Nµ²ªG¦C¦L¯È¤Wµe¤W¤@¾îªí¥Ü¶}©l³B, ¨Ã³]©w¯Èªº³t«×¬°0.5cm/min,
¦Ó§l¦¬«×ªº³æ¦ì«hÀ³¨Ì³J¥Õ½è¿@«×½Õ¾ã, 0.1©y©ó100pmol, 0.3©y©ó500pmol,
0.5©y©ó1nmol,1.0©y©ó2nmol, Yµ²ªGÅã¥Ü¦³¦Ã¬V¤§®pªi,
¥B¸g¤@¦b´úÅ秡µLªk®ø°£, «hbuffer©Îcolumn©Î¤GªÌ§¡n§ó´«¤F.
4.
±NSE³J¥Õ¼Ð·Ç²GÂ÷¤ß5min,
2,000xg, ©â¨ú100£gl´ú¸Õ,
¦pªG®p°ª¶W¹L¦C¦L¯È¤j¤p, ¥i½Õ¾ã¤§, Yµ²ªG¤ñvoid volumn¤j©ó2.5¿¥H¤W,
«hªí¥Ü³J¥Õ½è§lªþ¦bcolumn¤W.
5.
¨M©w¨CÓ®pªºelution
volume, ¦bø¥Hlog10¬°Áa¶b, ¦U®p¤§elution volumn(ml)¬°¾î¶bªº¹Ï,
¥H«K°µ´ú¥¼ª¾¼Ð¥»ªº°Ñ¦Ò.
6.
±µ¤U¨Ó®y¼Ð¥».
¤èªk¤ñ·Ó¹Lµ{3, 4.
SE
buffer°t¤è¡G
20mM
Sodium acetate, pH5.6
150mM
NaCl
SE
protein standards¡G
2.0mg
thyroglobulin
4.0mg
catalase
3.0mg
bovine serum albumin
3.0mg
ovalbumin
4.0mg
ribonuclease A
¦A¥[6ml
SE buffer¨Ã½w½w²V¦X, ¥²¶·§¹¥þ·»¸Ñ,
¨ÃÀ³¦bÅã·LÃè¤U¬d¬ÝµL¨I¾ý«á, ¦bÂ÷¤ß¤@¦¸, ¨ú¤W²M²G¨Ï¥Î.
ªþµù¡G
1.
SE HPLC ¼hªRªk, ©Ò±oªº¨CÓ³J¥Õ½èªºelution volume»P¦U³J¥Õ½èªºlog10(¤À¤l¶q)¦³Ãö.
2.
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3.
©Ò±o¤§µ²ªG¥i¦A¥H¹qªaªk¨Ó´ú¤À¤l¶q.
4.
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Toya
Soda SW
G
2,000 SW ¥Î©ó¤À¤l¶q30,000¥H¤U
G
3,000 SW ¥Î©ó¤À¤l¶q30,000¦Ü500,000
G
4,000 SW ¥Î©ó¤À¤l¶q500,000
.¤T¤Q¤».¥ÑBlot Transfer MembranesÀË´ú³J¥Õ½è
¦b¹qªa½¦¤¤ªº³J¥Õ½è, ¥i¥HÂಾ¦Ünitrocellalose½¤¤W, ¤Z¶q¡Ù50ngªº³J¥Õ½è±ø±a,
¥i¥H¥ÎIndia ink¬V¦â¦Ó§e¶Â¦â.
§÷
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0.3¢M(v/v)
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0.1¢MIndia
ink in Tween 20²G
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ªk¡G
1.
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¤@¦P©ñ¤J¶ì½¦²°¤¤, ¥HTween 20²G®ûªw, ¨Ã½wºC®¶Àú, ¦@¬~2¦¸,
¨C¦¸20¤ÀÄÁ.
2.
±N½¤¥HIndia
ink¬V¦â3hr, ©Î¹L©]¥ç¥i.
3.
±N½¤¥HTween
20²G¨R¬~, ¨Ãdestain(¥ç¥ÎTween 20²G), ¦Ü²M·¡¬°¤î.
¦A±N½¤¨ú¥X´½°®.
¤Wz¹Lµ{©|¥i³Ì¹w¥ý³B²z,
¥H¥[±j¬V¦â®ÄªG.
§÷
®Æ¡G
1¢MKOH
PBS
¤è
ªk¡G
1.
±N§t¦³³J¥Õ½èªº½¤©ñ¤J§t1¢MKOHªº¬Á¼þ½L¤¤,
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¥HPBS®û¬~2¦¸.
ªþµù¡G
¦b³B²z½¤®É§¡À³¥H§¨¤l³B²z, ¤Å¥H¤âª½±µ±µÄ².
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³J¥Õ½èSDS-PAGE(Polyacrylamide
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¦A¥Hantibody±´°wªþ¨ä¤W, ¦¹antibody¥i¥Hhorseradish peroxidase(HRPO)-IgªþµÛ,
¦A±N¦¹½¤®û¤J§@¥Î¦¨¤À¤¤¨ÏÅã²{µ²ªG.
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0.1¢MSDS
in PBS
Electroblotting
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Ponceau
S Solution
Blocking
buffer
Primary
antibody
Horseradish
peroxidase(HRPO)-anti-Ig conjugate
PBS
Diaminobenzidine(DAB)
substrate solution
Whatman
3MM filter paper
Scotch-Brite
pads(3M)
0.45£gm nitrocellulose
mambrane filter
Electroblotting
apparatus or Transflot appratus
Heat-sealable
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Photographic
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Plastic
box
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1.
º¥ý±N³J¥Õ½è¼Ð¥»¹qªa.
2.
¥HSDS-PAGE¹qªa¼Ð¥»(²Óµßµß¸s©Î·»¸Ñ«á¤§µß¸s¥i¥Hª½±µ¥[¤J0.1¢MSDS²G¤¤load¦Ügel¤W,ª`·Nn¦³¤@¤Õ©ñ³J¥Õ½è¤À¤l¶q¼Ð·Ç²G).(¥»¹êÅç«Ç¨Ï¥ÎªÌ¬°8¡Ñ10¡Ñ0.5cmªºmini
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3.
¸Ë³]Western
blot³]³Æ, ª`·NÀ¹¤â®M, ¥H§K¤â¤W¤â¯×¨Ï³J¥Õ½èµLªkBlot¦Ü½¤¤W.
¨ä¸Ë¸m¦p¤U¹Ï¥Ü¡G
¨Ï¥Î14V,
4¢J, 1~16hr¥i§¹¦¨. ¨Ï¥ÎElectroblotting buffer.
3.
±Nnitrocellulose
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2min, ±N¦¹µ²ªG·Ó¬Û.
4.
±N½¤©ñ¦b¶ì½¦³U¤º,
¥[¤Jblocking soluting(¨C10¡Ñ15cm2¤j¤pªº½¤¥Î5ml), ±N³U«Ê¤f,
½wºC®¶°Ê30min, ¦A¥´¶}³U¤f¤@¨¤, Ë¥h²GÅé.
5.
±NPrimary
antibodyµ}ÄÀ(³q±`monoclonal antibodiesµ}ÄÀ1¡G1,000, ¨ä¥L«hµ}ÄÀ1¡G100),
¦A¥H¨C10¡Ñ15cm2½¤¥Î5ml¨Ó®û, ¨Ã½wºC®¶°Ê30min.
6.
±N½¤¥Ñ³U¤¤¨ú¥X(À¹¤â®M),
©ñ¤J¶ì½¦¬Ö¤¤, ¥H200ml PBS¬~3¦¸, ¨C¦¸5min.
7.
µ}ÄÀHRPO
conjugate(¥Îblocking buffer).
8.
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¥H5ml HRPO conjugateµ}ÄÀ²G®û30min.
9.
Ë¥hHRPO
conjugate²G, ¨ú¥X½¤, ¥H200ml PBS¬~3¦¸, ¨C¦¸5min.
10.±N½¤¥H·í³õ°tªºDAB
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11.·Ó¬Û¦sÃÒ.
¦UºØ·»²G°t¤è¡G
Blocking
buffer
1g
§Y·»²æ¯×¥¤¯»ªw¤J100ml PBS¤¤
Diaminobenzidine
(DAB) substrate solution¡G
50ml
3,3-diaminobenzidine
2ml
1¢MCoCl2 in H2O
98ml
PBS
0.1ml
30¢MH2O2 (¦b¨Ï¥Î«e¤~¥[¤J)
(ª`·NDAB¦³PÀù©Ê,
n¤p¤ß¨Ï¥Î)
Electroblottiny
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±NpH½Õ¦Ü8.0, ¦A¥[1,200mlªºmethanol, ¦A¥HdH2O¨ÏÅé¿n¥[¦Ü6¤½¤É.
Poncean
S solution (0.5¢MPonceaus S, 1¢Macetic acid)¡G
0.5g
Ponceau S
1ml
glacial acetic acid
¥HdH2O¥[¦Ü100ml
Horseradish
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§¡¦V¼t°Ó±ÄÁÊ,
¨Ã¤À¸Ë0.025ml¾¯¶q«Ý¥Î.
ªþµù¡G
Western
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²¤¶¡G
¤HÅ餺ªº²ÓM¹J§Üì®É,
·|¨ü¨ë¿EÁc´Þ, ¦Ó²£¥Í¤j¶q¦³±M¤@©Êªº§ÜÅé(monoclonal antibody).
¦Ó§Ü¤¸±`¤Þ°_¤£¦PªºB²ÓM²£¥Í§ÜÅé,
¬G¦b¦å²G¤¤ªº§ÜÅé¹ê»Ú¤W¬O¦h¤¸©Ê§ÜÅé(polyclonal antibodies),
¨äì¦]¬O¦¹§Ü¤¸±`§t¦³¤£¥u¤@³BP§ÜÅ骺³¡¦ì(epitopes),
¨CÓ³¡¦ì³£¥i¤Þ°_¤@Ó¤£¦PB²ÓM§ÜÅé.
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(¦ý¦P¤@§ÜÅé¥u§t¨ä¤@), ¦ý«Áå«h¥u¦³¤@ºØ. §YIgG¬O£^, IgA¬O£\,
IgD¬O£_, IgM¬O£g, IgE¬O£`.
¤£½×»´,
«Á姡§t¦³Åܲ§ºÝ(variable region)»P«í±`ºÝ(constant region),
»P§Ü¤¸§@¥Îªº³¡¤À¬O¦b»´, «ÁåÅܲ§ºÝªº°ª«×Åܲ§(hypervariable
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¨Ã«D¤Hªº°ò¦]¦³¦p¦¹¤j¶q, ¦Ó¬O¦bB²ÓM¦¨¼ô¹Lµ{¤¤,
¨ä°ò¦]«²Õ(¦³«Ü¦h«í±`ºÝ, Åܲ§ºÝ, ³s±µºÝ, ÅܤÆdiversityºÝªº°ò¦],
¥i¥H«²Õ±Æ¦C), ¥i¥Hºc¦¨¤£¦Pªº§ÜÅé.
±N酶»Î±µ¦Ü§ÜÅ餧§Þ³N
±N酶»Î±µ¦Ü§ÜÅé¤W,
¬O¨Ï酶(¨Ò¦phorweradish peroxidase HRPO, ©Îalkaline phosphatase)»P§ÜÅé§Î¦¨Ã©wªº.
¥B¤£¼vÅT酶»P§ÜÅ餧¥\¯à.
¥HHRPO»Î±µ¬°¨Ò,
¨ä¤Æ¾ÇÅܤƦp¤U¡G
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¨ä¤èªk¦p¤U¡G
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1mg/ml
antibody solution
0.1M
phosphate buffer, pH6.8
Horseradish
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0.1M
Carbonate buffer, pH9.2
Sodium
periodate (NaIO4) solution, freshly prepared
Sodium
borohydride (NaBH4) solution, freshly prepared
Saturated
ammonium sulfate (NH4)2SO4 solution
Tris/EDTA/NaCl
(TEN) buffer, pH7.2
BSA
Glycerol
Dialysis
membrane
Pasteur
pipet fitted with glass wool
Sephadex
G-25, medium
¤è
ªk¡G
1.
±N1mg/ml§ÜÅé¥H2¤½¤É0.1M
phosphate buffer pH6.8³zªR, ¹L©],
4¢J ½w½wÅÍ©Õ. (§ÜÅé¶q¥i¥HA280/1.44¡×mg Ig/ml¨Ópºâ)
(³zªR½¤¼v 50¢Methanol®û¤@¤p®É,
¦A¥H10mM NaHCO3®û¤@¤p®É, ¦A¥H1mM EDTA®û¤@¤p®É, ¦A¥HdH2O¨R¬~,
¦A«O¦s¦bphosphaed buffer¤¤, 4¢J, ¬°¨¾²Óµß¥Íªø, buffer¤¤¥i¥[¤J0.01¢Mªºsodium
azide).
2.
·»10mgªºGRPO©ó1mlªº0.1M
carbonate buffer, pH9.2¤¤.
3.
±N0.25ml·s°t¦nªºNaIO4²G»P0.25mlªº10mg/ml
HRPO/carbonate²G²V¦X, »\ºò»\¤l, ¸m«Ç·Å2hr(ÁקK¥ú·Ó).
4.
¦b¤@¤äPasteur
pipette¤¤©ñ¤J¾A¶qglass wool, ±N¥X¤f³B¥Hparafilm¥]¦n, ¦A±N1ml¥H³zªR¹Lªºantibody²G,
¥[¤J0.5mlªº10mg/ml HRPO²G¤¤, ¦A±N0.25gªºSephadex G-25¥[¤J¦¹antebody/HRPO²V¦X²G¤¤
(¦¹¤@¨BÆJ¥i¼W¶i酶»P§ÜÅ餧µ²¦X). ±N¦¹²V¦Xª«¥[¤J¦¹Pasteur
pipette¤¤.
5.
¸m©ó·t«Ç,
«Ç·Å3hr.
6.
±N¦¹Column¥H0.75mlªºCarbonate
buffer±Nconjugate¬~¥X, «O¦s.
7.
¦b¦¹ÄÀ¥X²G¤¤,
¥[¤J38£glªº·s°t¦n¤§NaBH4²G, ¦b«Ç·Å, ·t«Ç,
¸m30min.
8.
¦A¥[¤J112£glªºNaBH4²G, ·t«Ç, ¸m60min.
9.
¦A¥[¤J¹¡©M0.9ml
(NH4)2SO4²G, ½w½wÅÍ©Õ30min, 4¢J, Â÷¤ß15min,
10,000xg.
10.Ë¥h¤W²M,
±N¨I¾ýª«·»©ó0.75mlªºTEN¤¤.
11.³zªR¦¹²G,
4¢J, 2¤½¤ÉªºTEN, ¹L©], ²Ä¤G¤Ñ´«¹L¤@¦¸TEN²G, ¦A³zªR4hr.
12.±N³zªR½¤¤ºª«¨ú¥X,
¥[¤J¾A¶qBSA, ¨Ï¦¹²G¤¤³Ì²×BSA¶q¬°20mg BSA/ml.
13.¥[¤Jµ¥¶q¤§glycerol¨Ã«O¦s¦b-20¢J.
°t¤è¡G
0.1M
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1.36g
Sodium carbonate
7.35g
Sodium bicarbonate
950ml
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¥H1M
HCl©Î1MªºNaOH½Õ¾ãpH¦Ü9.2¦A¥[dH2O¨ÏÅé¿n¦¨¬°1¤½¤É.
0.1M
phosphate buffer, pH6.8¡G
«O¦sA·»²G¡G0.2M¡G31.2g
NaH2PO4©ó1¤½¤ÉªºdH2O¤¤
«O¦sB·»²G¡G0.2M¡G28.39g
NaH2PO4©ó1¤½¤ÉªºdH2O¤¤
²V¦X51mlªºA²G»P49mlªºB²G,
¤Î100mlªºdH2O¦¨¬°¨Ï¥Î·»²G.
¹¡©Mammonium
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±N1.21g
Tris base¥[¤J990ml dH2O¤¤, ¨Ï¦¨0.01M Tris²G, ½Õ¾ãpH¦Ü7.0,
¦A¥[dH2O, ¨ÏÁ`Åé¿n¬°1¤½¤É, ¶q767gªº(NH4)2SO4¥[¤J¦¹Tris²G¤¤,ÅÍ©Õ,
¨Ã¥[¬°¼ö, ¨Ï·», ½Õ¾ãpH¦Ü7.0, ¨Ã«O¦s¦b4¢J, (NH4)2SO4ªºµ²´¹·|¨I¾ý¥X¨Ó.
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5mg
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Sodium
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1.71mg
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pH7.2¡G
¦b930ml dH2O¤¤¥[¤J¡G
0.06g
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8.77g
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¥[dH2O¨ÏÅé¿n¦¨¬°1¤½¤É
ªþµù¡G
1.
¨Ï¥Î¦¹ªk,
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2.
¦b¨Ï¥Î®É,
¥i±Nµ²ªGµ}ÄÀ100¦Ü10,000¿. ¬O®ÄªG¦p¦ó¦Ó©w.
·Ç³Æ²Óµß§Ü¤¸
§÷
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E.Coli(ªø©ó°ö¾i²G¤¤)
Cell
resuspending buffer
Lysozyme
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Tris/EDTA/NaCl(TEN)
buffer
10¢MSDS
8M
urea
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ªk¡G
1.
±N5mlªºE.Coli²G¦b®à±Â÷¤ß¾÷Â÷¤ß2,500rpm,
10min, Ë¥h¤W²M, ±N¨I¾ý²Óµß·»©ó5ml resuspending buffer¤¤,
¥R¤À²V¦X(Votex).
2.
§l¥X1ml²Óµß²G¤J1.5mlªº·L¶qÂ÷¤ßºÞ¤¤,
¸m¦B¤W¦A¥[¤J0.2mlªºlysozyme²G, «Ý5min.
3.
Â÷¤ß3,000rpm,
5min, «O¯d¤W¼h, ±N¨I¾ýª«¥H1.2ml TEN·»¸Ñ¤§. (¦]¬°¦³¨Ç³J¥Õ½è¬O¤£·»©ó¤ô,
¬G¤GªÌ§¡À³«O¯d)
4.
¦b¨C¤@ºØ¼Ë¥»¤¤¦U¥[¤J65£glªº10¢MSDS,
¸m37¢J, 10min, ¦¹®É¼Ë¥»§Y¥i¨Ï¥Î, Y¤£¨Ï¥ÎÀ³§Ná«O¦s,
Y¦³¥²n¥i¥H8M urea²G(4.8g urea¥[¤J10ml²G¤¤), ³B²z³J¥Õ½è.
°t¤è¡G
cell
resuspending buffer (10mM HEPES)
2.38g
HEPES
¥[dH2O©Î1
liter
ªþµù¡G
1.
¦b§K¬Ì¾Ç¹êÅ礤,
Enzyme-Linked Immunolorbent Assays(ELISA)¬O±`¥Î©ó°»´ú²Óµß©Î§Ü¤¸ªº¤èªk.
¤@¯ë¤À¬°ª½±µªk, §Y±N§Ü¤¸©ñ¤Jmicro titer plate, ¨ÏªþµÛ¦b¤º¾À,
¦A¥Hdiluting buffer¨Ï¥¼³QªþµÛ³B¯à¤£±µ¨ü§ÜÅéªþµÛ,
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¦A¥[¤J§Üì©l»P§ÜÅé§@¥Î, ¦A¬~¥h¦h¾lªº§ÜÅé,
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³\¦h°ò¦]¥\¯à±±¨î¨ü¨ì»P¦¹°ò¦]¤¤¬Y¤@¤ùÂ_»P¬Y¯S®í³J¥Õ½èªºµ²¦X¤§¼vÅT,
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¨C¤½¤É§t10g
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7. ¨C¤ä¸ÕºÞ¥[¤J0.7ml PEG²G, ¥H¤â«ü»´¼u¸ÕºÞ²V¦X¤§, ¸m30¢J, 45min, ¦A¸m42¢J, 5min.
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