เอกสารการประชุมวิชาการและเสนอผลงานวิจัย มหาวิทยาลัยทักษิณ ครั้งที่ 19 2552 - page 316

„µ¦ž¦³»¤ª·µ„µ¦Â¨³Áœ°Ÿ¨Šµœª·‹´¥ ¤®µª·š¥µ¨´¥š´„¬·– ‡¦´ÊŠš¸É 19 ž¦³‹Îµže 2552
œ°µ®µ¦Â Ȋ Luria Bertani (LB) š¸É¤¸¥µž’·¸ªœ³ ampicillin ‡ªµ¤Á o¤ oœ 100 mg/ml š¸É°»–®£¼¤· 37qC Áž} œÁª¨µ
16 ´ÉªÃ¤Š ‡´—Á¨º°„Ç蜸š¸É ¤¸¦¸‡°¤·Âœœšr—¸Á°ÈœÁ° °Š pSUMO-Hb REF —oª¥Áš‡œ·‡ PCR ¨³ª·Á‡¦µ³®r®µ¨Îµ—´
ÁÃ—¥ÄoÅ¡¦Á¤°¦r‹ÎµÁ¡µ³„´ pSUMO vector (5'-GAAGATTTGGACATGGAGGAT-3')
„µ¦´ŠÁ‡¦µ³®r¦¸‡°¤·ÂœœšrÞ¦˜¸ œ HbREF Ĝ E. coli BL21 plus (DE3) RIL
ª·Á‡¦µ³®r‡ªµ¤Á o¤ oœ °Š IPTG š¸ÉÁ®¤µ³¤
œÎ µÁºÊ° E. coli BL21 plus (DE3) RIL š¸É¤¸¡¨µ¤· — pSUMO-REF Á¨¸Ê¥ŠÄœ°µ®µ¦Á®¨ª LB ž¦·¤µ˜¦ 1 ml š¸É
Ÿ¤¥µž’·¸ªœ³ ampicillin ‡ªµ¤Á o¤ oœ 100 mg/mL š¸É °»–®£¼¤· 37 qC Áž}œÁª¨µ 16 ´ÉªÃ¤Š Á¡ºÉ°ÄoÁž}œÁŽ¨¨rÁ¦·É¤˜oœ
‹µ„œ´Ê œœÎµÁŽ¨¨rÁ¦·É ¤˜oœ ž¦· ¤µ˜¦ 400 Pl Á¨¸Ê¥ŠÄœ°µ®µ¦Á®¨ª LB ž¦·¤µ˜¦ 4 ml š¸É¤¸ n ªœŸ¤ °Š¥µž’·¸ªœ³
ampicillin ‡ªµ¤Á o¤ oœ 100 mg/mL š¸É °»–®£¼¤· 37qC Áž} œÁª¨µ 3 ´ÉªÃ¤Š (OD600=0.6) ‹µ„œ´ÊœÂnŠ°µ®µ¦Á¨¸Ê¥ŠÁºÊ°
°°„Áž} œ 4 ®¨°— ®¨°—¨³ 1 ml ×¥®¨°—š¸É 1 Ťn¤¸„µ¦Á®œ¸É¥ªœÎ µ (uninduced) Ä®o¤¸„µ¦¦oµŠÃž¦˜¸ œ Hb REF ‹¹ŠÅ¤n
Á˜·¤ isopropyl O-D-thiogalactopyronoside (IPTG ) Î µ®¦´®¨°—š¸É 2-4 Á®œ¸É ¥ªœÎµ (induced) Ä®o¤¸„µ¦¦oµŠÃž¦˜¸œ
HbREF ×¥Á˜·¤ IPTG š¸É‡ªµ¤Á o¤ oœ 0.05 mM, 0.1 mM ¨³ 0.2 mM ˜µ¤¨Îµ—´ ‹µ„œ´ÊœÁ¨¸Ê ¥ŠÁºÊ°Åªoš¸É °»–®£¼¤·
37qC Áž} œÁª¨µ 12 ´ÉªÃ¤Š œÎ µÁºÊ°¤µ®¤»œÁ®ª¸É¥Šš¸É‡ªµ¤Á¦Èª 10,000xg Áž}œÁª¨µ 10 œµš¸ ‹µ„œ´ÊœÁ˜¦¸¥¤Ãž¦˜¸ œ—oª¥ª·›¸
freeze-thaw lysis ×¥œÎµ˜³„°œÁºÊ ° E .coli BL21 plus (DE3) RIL š´ÊŠ uninduced ¨³ induced —oª¥ IPTG ¨³¨µ¥
—oª¥µ¦¨³¨µ¥ STE (0.1M NaCl, 50mM Tris-HCl pH6.8, 1 mM EDTA) ž¦·¤µ˜¦100 Pl š¸ÉŸ¤—o ª¥ 1 mM PMSF,
1 mg/ml lysozyme, 5U DNase ¨³ 10 mg/ml RNase ªµŠÅªoš¸É °»–®£¼¤·®o °ŠÁž}œÁª¨µ 30 œµš¸ ‹µ„œ´Êœ‹¹ŠÁ„È Åªoš¸É
°»–®£¼¤· -20qC Áž}œÁª¨µ 3 ´ÉªÃ¤Š ¨³œÎµ°°„¤µªµŠš¸É°»–®£¼ ¤·®o°Š‹œµ¦¨³¨µ¥˜³„°œÁºÊ °¨³¨µ¥ n¤Äœ„pµŽ
ŜئÁ‹œÁ®¨ªÁž}œÁª¨µ 1 œµš¸ ¨³——oª¥ glass Potter-Elvehjem homogenizer ¨oª®¤»œÁ®ª¸É¥Šš¸É‡ªµ¤Á¦Èª 10,000
x g Áž}œÁª¨µ 15 œµš¸ Á„ȝµ¦¨³¨µ¥Ãž¦˜¸ œÎµ®¦´ ª·Á‡¦µ³®r Þ¦˜¸œš¸É¨³¨µ¥ (soluble protein) 宦´ ˜³„°œÁºÊ°
Á˜·¤ SDS loading buffer (0.35M Tris-HCl pH 6.8, 10.28% SDS, 36% glycerol, 5% E-mercaptoethanol ¨³
0.012% bromophenol blue) ž¦· ¤µ˜¦ 50 Pl ¨oª˜o¤š¸É °»–®£¼¤· 95 qC Áž}œÁª¨µ 15 œµš¸ ®¤» œÁ®ª¸É¥Šš¸É‡ªµ¤Á¦Èª 10,000
x g Áž}œÁª¨µ 15 œµš¸ Á„ȝnªœÄ´Êœœ (supernatant) ‹µ„œ´Êœ‹¹ŠœÎµµ¦¨³¨µ¥Ãž¦˜¸œš¸ÉÁ˜¦¸¥¤Å—oޚε SDS-PAGE
(acrylamide:bisacrylamide; 29:1) š¸É ‡ªµ¤Á o¤ oœ °Š polyacrylamide gel ´Êœœ (stacking) 4% ¨³´Ê œ¨nµŠ
(separating) 15% ‹µ„œ´Êœ¥o °¤Â™Ãž¦˜¸œ—oª¥ 0.2% Coomassie blue R250 (¨³¨µ¥Äœ 40% methanol ¨³10%
acetic acid)
ª·Á‡¦µ³®r°»–®£¼¤· n¤ÁŽ¨¨rš¸É Á®¤µ³¤
œÎ µÁºÊ°Á¦·É¤˜oœž¦·¤µ˜¦ 400 Pl Á¨¸Ê¥ŠÄœ°µ®µ¦Á®¨ª LB ž¦· ¤µ˜¦ 4 ml š¸É¤¸¥µž’· ¸ªœ³ ampicillin ‡ªµ¤
Á o¤ oœ 100 mg/mL š¸É°»–®£¼¤· 37qC Áž} œÁª¨µ 3 ´ÉªÃ¤Š (OD600=0.6) ‹µ„œ´ÊœÂnŠ°µ®µ¦Á¨¸Ê ¥ŠÁºÊ°°°„Áž}œ 4 ®¨°—
®¨°—¨³ 1 ml ×¥®¨°—š¸É 1 ŤnÁ˜·¤ IPTG (uninduced) ®¨°—š¸É 2-4 induced —oª¥ IPTG š¸É ‡ªµ¤Á o¤ oœ 0.1 mM
‹µ„œ´ÊœÁ¨¸Ê ¥ŠÁºÊ°Åªo š¸É°»–®£¼¤· 15qC, 25qC ¨³ 37qC Áž} œÁª¨µ 12 ´ÉªÃ¤Š ®¤»œÁ®ª¸É ¥Šš¸É‡ªµ¤Á¦Èª 10,000xg Áž}œÁª¨µ
10 œµš¸ ¨³Á„ȝ˜³„°œÁºÊ°Åªo š¸É°»–®£¼¤· -20qC ‹µ„œ´Êœ‹¹ŠÁ˜¦¸¥¤Ãž¦˜¸ œ‹µ„nªœ˜nµŠÇ ¨³˜¦ª‹°Ãž¦˜¸œ—oª¥
SDS-PAGE ˜µ¤ª·›¸„µ¦š¸É„¨nµªÄœ˜°œ˜oœ
ª·Á‡¦µ³®r¦³¥³Áª¨µn¤ÁŽ¨¨rš¸ÉÁ®¤µ³¤
œÎ µÁºÊ°Á¦·É¤˜oœž¦·¤µ˜¦ 400 Pl Á¨¸Ê¥ŠÄœ°µ®µ¦Á®¨ª LB ž¦· ¤µ˜¦ 4 ml š¸É¤¸¥µž’· ¸ªœ³ ampicillin ‡ªµ¤
Á o¤ oœ 100 mg/mL š¸É°»–®£¼¤· 37qC Áž} œÁª¨µ 3 ´ÉªÃ¤Š (OD600=0.6) ‹µ„œ´ÊœÂnŠ°µ®µ¦Á¨¸Ê ¥ŠÁºÊ°°°„Áž}œ 4 ®¨°—