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Method for extracting protein from tissue or cell

ย  1. Introduction

“Protein”์˜ ์–ด์›์€ ๊ทธ๋ฆฌ์„œ์–ด์˜ “Proteios (๊ฐ€์žฅ ์ค‘์š”ํ•œ ๊ฒƒ)”์—์„œ ์œ ๋ž˜ํ–ˆ๋‹ค๊ณ  ํ•ฉ๋‹ˆ๋‹ค. ๋‹จ๋ฐฑ์งˆ์€ ์ƒ์ฒด์˜ ๊ตฌ์กฐ์™€ ๊ธฐ๋Šฅ์„ ๋‹ด๋‹นํ•˜๋Š” ๋ฐ ํ•„์ˆ˜์ ์ธ ์กด์žฌ๋ผ๊ณ  ํ•  ์ˆ˜ ์žˆ์Šต๋‹ˆ๋‹ค. ๋‹จ๋ฐฑ์งˆ์˜ ๊ธฐ๋Šฅ์„ ์—ฐ๊ตฌํ•˜๊ธฐ ์œ„ํ•ด์„œ๋Š” ๋จผ์ €, ์ƒ์ฒด ์ƒ˜ํ”Œ์—์„œ ๋‹จ๋ฐฑ์งˆ์„ ์ถ”์ถœํ•˜์—ฌ ๊ฐ€์šฉํ™”ํ•˜๋Š” ๊ฒƒ์ด ์ค‘์š”ํ•ฉ๋‹ˆ๋‹ค. ์—ฌ๊ธฐ์—์„œ๋Š” ์•„ํ† ์˜ ์ œํ’ˆ์„ ์‚ฌ์šฉํ•˜์—ฌ ๋‹จ๋ฐฑ์งˆ์„ ์ถ”์ถœํ•˜๋Š” ๋ฐฉ๋ฒ•์— ๋Œ€ํ•ด ์†Œ๊ฐœํ•˜๊ฒ ์Šต๋‹ˆ๋‹ค.

โ–ถ ์ฃผ์˜ํ•  ์ 

๋‹จ๋ฐฑ์งˆ์„ ์ถ”์ถœํ•  ๋•Œ ๊ธฐ๋ณธ์ ์ธ ์ฃผ์˜์ ์œผ๋กœ๋Š”
1. ์ƒ˜ํ”Œ ์„ ํƒ (์‹ ์„ ํ•˜๊ณ  ์›ํ•˜๋Š” ๋ชฉ์ ์„ ์•Œ๊ธฐ ์‰ฌ์šด ์ƒ˜ํ”Œ ์„ ํƒ)
2. ์˜จ๋„ (์ผ๋ฐ˜์ ์œผ๋กœ 0~4โ„ƒ)
3. pH, ์ด์˜จ๊ฐ•๋„ (ionic strength): pH๋Š” ์ผ๋ฐ˜์ ์œผ๋กœ pH 4~7, ์ด์˜จ๊ฐ•๋„๋Š” 0.1M
4. Thiol๊ธฐ (์‚ฐํ™”๋ฅผ ๋ณดํ˜ธํ•˜๊ธฐ ์œ„ํ•œ DTT ๋“ฑ์˜ ํ™˜์›์ œ ์ฒจ๊ฐ€)
5. ์•ˆ์ •ํ™”์ œ์˜ ์ฒจ๊ฐ€ (๋‹น์ด๋‚˜ ๊ธ€๋ฆฌ์„ธ๋ฆฐ ๋“ฑ์˜ ์ฒจ๊ฐ€)
6. Protease inhibitor์˜ ์ฒจ๊ฐ€
7. ๊ธˆ์†์ด์˜จ chelate ๋“ฑ์ด ์žˆ์Šต๋‹ˆ๋‹ค.

ย  2. ๋Œ€์žฅ๊ท ์˜ ๋‹จ๋ฐฑ์งˆ ์ถ”์ถœ ๋ฐฉ๋ฒ•

๋Œ€์žฅ๊ท ๊ณผ ๊ฐ™์€ ๋ฏธ์ƒ๋ฌผ์—๋Š” ์„ธํฌ๋ฒฝ์ด ์žˆ๊ธฐ ๋•Œ๋ฌธ์— Mammalian cell์— ๋น„ํ•ด ํŒŒ๊ดดํ•˜๋Š” ๊ฒƒ์ด ์–ด๋ ต์Šต๋‹ˆ๋‹ค. ์ผ๋ฐ˜์ ์œผ๋กœ๋Š” ์ดˆ์ŒํŒŒ ํŒŒ์‡„, ๋™๊ฒฐ ์œตํ•ด๋ฅผ ํ†ตํ•œ ํŒŒ์‡„, Lysozome๋“ฑ์˜ ํšจ์†Œ์— ์˜ํ•œ ์šฉํ•ด ๋“ฑ์„ ํ†ตํ•ด ๋‹จ๋ฐฑ์งˆ์„ ์ถ”์ถœํ•ฉ๋‹ˆ๋‹ค. ๊ทธ๋Ÿฌ๋‚˜ ์ดˆ์ŒํŒŒ๋‚˜ ๋™๊ฒฐ ์œตํ•ด์— ์˜ํ•œ ๋ฌผ๋ฆฌ์  ํŒŒ์‡„๋Š” ์กฐ์ž‘์ด ๋ฒˆ๊ฑฐ๋กญ๊ณ  ์ข…์ข… ๋‹จ๋ฐฑ์งˆ์ด ๋ณ€์„ฑ๋˜๋Š” ์›์ธ์ด ๋ฉ๋‹ˆ๋‹ค. ATTO์˜ WSE-7423 EzBactYeastCrusher๋Š” ๊ท ์ฒด์— ๋„ฃ๊ณ  ์„ž๋Š” ๊ฒƒ๋งŒ์œผ๋กœ๋„ ๋‹จ๋ฐฑ์งˆ์„ ์•ˆ์ •์ ์œผ๋กœ ์ถ”์ถœํ•  ์ˆ˜ ์žˆ์Šต๋‹ˆ๋‹ค.

2-1. ์‹คํ—˜๋ฐฉ๋ฒ•

์‹คํ—˜์žฌ๋ฃŒ:
๋Œ€์žฅ๊ท  ๋ฐ ๋ฐฐ์–‘์•ก
WSE-7423 EzBactYeastCrusher
์ฆ๋ฅ˜์ˆ˜
๋งˆ์ดํฌ๋กœ ์›์‹ฌํŠœ๋ธŒ, ํ”ผํŽซ tip ๋“ฑ
์›์‹ฌ๊ธฐ, ๋งˆ์ดํฌ๋กœํ”ผํŽซ ๋“ฑ

๋Œ€์žฅ๊ท  ๋ฐฐ์–‘
๋Œ€์žฅ๊ท ์„ ์ ๋‹นํ•œ ๋ฐฐ์ง€์—์„œ O.D. 600=0.5~1.0์ด ๋ ๋•Œ๊นŒ์ง€ ๋ฐฐ์–‘ํ•ฉ๋‹ˆ๋‹ค. (๊ท ์ฒด๋Ÿ‰์œผ๋กœ๋Š” 50~100 mg ์ƒ๋‹น)
* ๋Œ€์žฅ๊ท  ๋ฐœํ˜„ ๋‹จ๋ฐฑ์งˆ์„ ์ถ”์ถœํ•˜๋Š” ๊ฒฝ์šฐ์—๋Š” ๋ฐœํ˜„์šฉ ๋Œ€์žฅ๊ท  ๋ฐ ๋ฐœํ˜„ ๋‹จ๋ฐฑ์งˆ์— ์ ์ ˆํ•œ ์กฐ๊ฑด์—์„œ ๋ฐฐ์–‘ํ•  ๊ฒƒ์„ ๊ถŒ์žฅํ•ฉ๋‹ˆ๋‹ค.

Collect, Washing
๋Œ€์žฅ๊ท ์„ 2,000ร—g๋กœ 5๋ถ„๊ฐ„ ์›์‹ฌ๋ถ„๋ฆฌํ•˜์—ฌ ์ƒ๋“ฑ์•ก์„ ์ œ๊ฑฐํ•ฉ๋‹ˆ๋‹ค. Pellet์— 5~10mL์˜ ์ฆ๋ฅ˜์ˆ˜๋ฅผ ๋”ํ•ด ํ˜„ํƒํ•˜์—ฌ ๋‹ค์‹œ 2,000ร—g์—์„œ 5๋ถ„๊ฐ„ ์›์‹ฌ๋ถ„๋ฆฌํ•˜์—ฌ ์ƒ๋“ฑ์•ก์„ ์ œ๊ฑฐํ•ฉ๋‹ˆ๋‹ค.
* Washingํ›„์˜ Pellet (๊ท ์ฒด)๋Š” -80โ„ƒ์—์„œ ๋™๊ฒฐ๋ณด์กด ๊ฐ€๋Šฅํ•ฉ๋‹ˆ๋‹ค. ์ผ๋ฐ˜์ ์œผ๋กœ ํ•œ ๋ฒˆ ๋™๊ฒฐํ•˜๋ฉด ์ถ”์ถœ ํšจ์œจ์ด ๋†’์•„์ง‘๋‹ˆ๋‹ค.

๊ท ์ฒด ์šฉํ•ด
์ƒ๋“ฑ์•ก์„ ์ œ๊ฑฐํ•˜๊ณ  ๊ท ์ฒด๋ฅผ vortexํ•˜์—ฌ ์„ธํฌ๋ฅผ ์ž˜ ํ’€์–ด์ค๋‹ˆ๋‹ค. ์ด ๋•Œ ๊ท ์ฒด๋ฅผ ๊ท ์ผํ•˜๊ฒŒ ์ž˜ ํ’€์–ด์ฃผ๋Š” ๊ฒƒ์ด ์ค‘์š”ํ•ฉ๋‹ˆ๋‹ค.
0.5 mL์˜ EzBactYeast Lysis buffer๋ฅผ ์ฒจ๊ฐ€ํ•˜์—ฌ ์‹ค์˜จ์— 10๋ถ„๊ฐ„ ๋‘ก๋‹ˆ๋‹ค.
* EzBactYeast Lysis buffer์— ๋ฏธ๋ฆฌ EzBactYeast Crusher ํ‚คํŠธ์— ํฌํ•จ๋œ Protease Inhibitor์™€ DNase I์„ ์ฒจ๊ฐ€ํ•ฉ๋‹ˆ๋‹ค.

๋‹จ๋ฐฑ์งˆ Extraction
๊ท ์ฒด์šฉํ•ด์•ก์„ 4โ„ƒ, 10,000ร—g์—์„œ 5๋ถ„๊ฐ„ ์›์‹ฌ๋ถ„๋ฆฌํ•˜์—ฌ ์ƒ๋“ฑ์•ก์„ ๋ชจ์•„์ค๋‹ˆ๋‹ค.
* ๋ถˆ์šฉ์„ฑ ๋‹จ๋ฐฑ์งˆ์€ Pellet์— ํšŒ์ˆ˜๋ฉ๋‹ˆ๋‹ค. ๋ถˆ์šฉ์„ฑ ๋‹จ๋ฐฑ์งˆ์„ ์ถ”์ถœํ•  ๋•Œ์—๋Š” Pellet์— 8M Urea contained buffer๋‚˜ 6M Guanidine-HCl contained buffer ๋“ฑ์„ ์ฒจ๊ฐ€ํ•ด ์šฉํ•ดํ•˜์—ฌ ๋‹จ๋ฐฑ์งˆ์„ ์ถ”์ถœํ•˜์—ฌ ์ฃผ์‹ญ์‹œ์˜ค.

์ผ๋ฐ˜์ ์ธ ๋Œ€์žฅ๊ท  ๋‹จ๋ฐฑ์งˆ ์ถ”์ถœ๋ฐฉ๋ฒ•

1. ๋Œ€์žฅ๊ท ์„ ํŒŒ์‡„์šฉ ์šฉ์•ก(50 mM Tris pH8.0, 500 mM NaCl, 15% glycerol, 10 mM imidazole)์œผ๋กœ 3ํšŒ ์„ธ์ฒ™ํ•œ๋‹ค.
2. Pellet์„ 3๋ฐฐ ์šฉ๋Ÿ‰์˜ Sonication buffer์— ํ˜„ํƒํ•˜๊ณ , ์ตœ์ข…๋†๋„๊ฐ€ 1 mg/mL Lysozyme, 1 mM DTT, 0.5 mM EDTA๊ฐ€ ๋˜๋„๋ก ์ฒจ๊ฐ€ํ•œ๋‹ค.
3. ๋Œ€์žฅ๊ท ์ด ํŒŒ์‡„๋  ๋•Œ๊นŒ์ง€ ๋ƒ‰๊ฐํ•˜๋ฉด์„œ ์ดˆ์ŒํŒŒ ํŒŒ์‡„๋ฅผ ์‹ค์‹œํ•œ๋‹ค (ํ˜„๋ฏธ๊ฒฝ ๋“ฑ์œผ๋กœ ํ™•์ธ).
4. DNase I์˜ ์ตœ์ข…๋†๋„๊ฐ€ 0.1 mg/mL์ด ๋˜๋„๋ก ์ฒจ๊ฐ€ํ•œ๋‹ค.
5. ์›์‹ฌ๋ถ„๋ฆฌ(10,000ร—g~25,000ร—g, 10~30๋ถ„)ํ•˜์—ฌ ์ƒ๋“ฑ์•ก์„ ํšŒ์ˆ˜ํ•œ๋‹ค.

2-2. ์ฐธ๊ณ  ๋ฐ์ดํ„ฐ

์‹คํ—˜์˜ˆ1: ์ถ”์ถœ ํšจ์œจ

์ถ”์ถœ์— ์‚ฌ์šฉํ•œ ์‹œ์•ฝ

A: WSE-7423 EzBactYeastCrusher (์‹ค์˜จ)

C: ํƒ€์‚ฌ ์‹œํŒ ์‹œ์•ฝ

B: ํƒ€์‚ฌ ์‹œํŒ ์‹œ์•ฝ

L: Lysozyme

S: SDS sample buffer

A: WSE-7423 EzBactYeastCrusher (4โ„ƒ)

M: EzProtein Ladder

Luciferase ๋ฐœํ˜„ ๋Œ€์žฅ๊ท ์—์„œ ์œ„์— ๊ธฐ์žฌ๋œ ์‹œ์•ฝ์„ ์‚ฌ์šฉํ•˜์—ฌ ๋‹จ๋ฐฑ์งˆ์„ ์ถ”์ถœํ•˜๊ณ , ์ „๊ธฐ์˜๋™ํ•œ ๊ฒฐ๊ณผ (์œ„)์™€ ํšจ์†Œ ํ™œ์„ฑ์„ ์ธก์ •ํ•œ ๊ฒฐ๊ณผ (์™ผ์ชฝ)์„ ๋‚˜ํƒ€๋ƒˆ์Šต๋‹ˆ๋‹ค.ย WSE-7423 EzBactYeastCrusher๋Š” ์‹ค์˜จ์—์„œ๋„, ์ €์˜จ(4โ„ƒ)์—์„œ๋„ ๋™์ผํ•˜๊ฒŒ ํƒ€์‚ฌ ์‹œํŒ ์‹œ์•ฝ๊ณผ ๋™๋“ฑ ์ด์ƒ์˜ ์ถ”์ถœ ํšจ์œจ๋กœ ๋‹จ๋ฐฑ์งˆ์„ ํšŒ์ˆ˜ํ•  ์ˆ˜ ์žˆ์Šต๋‹ˆ๋‹ค.

๋˜ํ•œ ์™ผ์ชฝ์˜ ๊ทธ๋ž˜ํ”„์— ์ œ์‹œ๋œ ๋ฐ”์™€ ๊ฐ™์ด ์‹ค์˜จ์—์„œ ์ถ”์ถœํ•œ ๊ฒฝ์šฐ์—๋„ ํšจ์†Œ ํ™œ์„ฑ์„ ๋†’๊ฒŒ ์œ ์ง€ํ•œ ์ƒํƒœ์—์„œ ๋‹จ๋ฐฑ์งˆ ์ถ”์ถœ์ด ๊ฐ€๋Šฅํ•จ์„ ์•Œ ์ˆ˜ ์žˆ์Šต๋‹ˆ๋‹ค.

์‹คํ—˜์˜ˆ2: ๋ฐœํ˜„ ๋‹จ๋ฐฑ์งˆ์˜ ์ •์ œ

๊ทธ๋ฆผ์€ His tag-Luciferase ๋ฐœํ˜„ ๋Œ€์žฅ๊ท ์—์„œ WSE-7423 EzBactYeastCrusher๋ฅผ ์‚ฌ์šฉํ•˜์—ฌ ์ถ”์ถœํ•œ ๋‹จ๋ฐฑ์งˆ์„ Ni-Sepharose ์ปฌ๋Ÿผ์œผ๋กœ ์ •์ œํ•œ ๊ฒฐ๊ณผ๋ฅผ ๋‚˜ํƒ€๋ƒˆ์Šต๋‹ˆ๋‹ค. WSE-7423 EzBactYeastCrusher๋Š” ์ปฌ๋Ÿผ์œผ๋กœ์˜ ๊ฒฐํ•ฉ, ๊ฐ ์ •์ œ ์Šคํ…์˜ ๋ฐ˜์‘์„ ์ €ํ•ดํ•˜์ง€ ์•Š์œผ๋ฉฐ ํšจ์†Œ ํ™œ์„ฑ๋„ ์œ ์ง€ํ•œ ์ƒํƒœ์—์„œ ๋‹จ๋ฐฑ์งˆ์„ ์ •์ œํ•  ์ˆ˜ ์žˆ์Šต๋‹ˆ๋‹ค. ์ด์™€ ๊ฐ™์ดWSE-7423 EzBactYeastCrusher๋Š” ๋Œ€์žฅ๊ท ๊ณ„ ๋ฐœํ˜„ ๋‹จ๋ฐฑ์งˆ์˜ ์ถ”์ถœ ๋ฐ ์ •์ œ์—๋„ ์ ํ•ฉํ•œ ๊ฒƒ์œผ๋กœ ๋‚˜ํƒ€๋‚ฌ์Šต๋‹ˆ๋‹ค.

M: WSE-7020 EzProtein Ladder
Ext: Whole Extract
FT: Flow through
W: Wash
E1: Extraction 1
E2: Extraction 2 (Purified protein)

ย  3. ํšจ๋ชจ์˜ ๋‹จ๋ฐฑ์งˆ ์ถ”์ถœ ๋ฐฉ๋ฒ•

ํšจ๋ชจ์—๋Š” ๋‹ค๋‹น๋ฅ˜๋ฅผ ์ฃผ์„ฑ๋ถ„์œผ๋กœ ํ•œ ๊ฒฝ์งˆ ์„ธํฌ๋ฒฝ์ด ์žˆ๊ธฐ ๋•Œ๋ฌธ์— ๋Œ€์žฅ๊ท ๋ณด๋‹ค ํŒŒ๊ดดํ•˜๋Š” ๊ฒƒ์ด ์–ด๋ ต์Šต๋‹ˆ๋‹ค. ์ผ๋ฐ˜์ ์œผ๋กœ๋Š” Glass beads ๋“ฑ์˜ ์—ฐ๋งˆ์ œ๋ฅผ ์ฒจ๊ฐ€ํ•œ Homogenizer, French press ๋“ฑ์˜ ๊ฐ€์••๊ฐ์••์ฒ˜๋ฆฌ์— ์˜ํ•œ ํŒŒ์‡„, ์ดˆ์ŒํŒŒ ํŒŒ์‡„, Zymolyase๋“ฑ์˜ ํšจ์†Œ์— ์˜ํ•ด Protoplastํ™”๋œ ์ดํ›„์˜ ์šฉํ•ด ๋“ฑ์ด ์ฃผ๋ฅ˜์ž…๋‹ˆ๋‹ค. ๊ทธ๋Ÿฌ๋‚˜ ๋ฌผ๋ฆฌ์  ํŒŒ๊ดด๋Š” ์ถ”์ถœ ํšจ์œจ์€ ๋†’์ง€๋งŒ ์กฐ์ž‘์ด ๋ฒˆ์žกํ•˜๊ณ  ๋ฌผ๋ฆฌ์  ์†์ƒ์ด ํฌ๋ฉฐ, ๋˜ Protoplastํ™”๋„ ์กฐ๊ฑด์˜ ์ตœ์ ํ™”๊ฐ€ ์–ด๋ ต๋‹ค๊ณ  ํ•ฉ๋‹ˆ๋‹ค. ATTO์˜ WSE-7423 EzBactYeastCrusher๋Š” Protease Inhibitor์™€ DNase I์ด ํฌํ•จ๋˜์–ด ์žˆ์–ด ๊ท ์ฒด์— ์„ž๋Š” ๊ฒƒ๋งŒ์œผ๋กœ๋„ Glass beads๋ฒ•๊ณผ ์œ ์‚ฌํ•œ ์ถ”์ถœํšจ์œจ๋กœย  ๋‹จ๋ฐฑ์งˆ์„ ์•ˆ์ •์ ์œผ๋กœ ์ถ”์ถœํ•  ์ˆ˜ ์žˆ์Šต๋‹ˆ๋‹ค. (Schizosaccharomyces pombe๋Š” ์ถ”์ถœํšจ์œจ์ด ๋–จ์–ด์ง‘๋‹ˆ๋‹ค.)

3-1. ์‹คํ—˜๋ฐฉ๋ฒ•

์‹คํ—˜์žฌ๋ฃŒ
๊ท ์ฒด ๋ฐ ๋ฐฐ์–‘์•ก (10 mL)
WSE-7423 EzBactYeastCrusher
์ฆ๋ฅ˜์ˆ˜
๋งˆ์ดํฌ๋กœ ์›์‹ฌํŠœ๋ธŒ, ํ”ผํŽซ tip ๋“ฑ
์›์‹ฌ๊ธฐ, ๋งˆ์ดํฌ๋กœํ”ผํŽซ ๋“ฑ

ํšจ๋ชจ ๋ฐฐ์–‘
ํšจ๋ชจ๋ฅผ ์ ๋‹นํ•œ ๋ฐฐ์ง€์—์„œ O.D.600=0.5~1.0์ด ๋ ๋•Œ๊นŒ์ง€ ๋ฐฐ์–‘ํ•ฉ๋‹ˆ๋‹ค. (๊ท ์ฒด๋Ÿ‰์œผ๋กœ๋Š” 50~100 mg ์ƒ๋‹น)
* ๋ฐœํ˜„ ๋‹จ๋ฐฑ์งˆ์„ ์ถ”์ถœํ•  ๊ฒฝ์šฐ์—๋Š” ๋ฐœํ˜„์šฉ ํšจ๋ชจ ๋ฐ ๋ฐœํ˜„ ๋‹จ๋ฐฑ์งˆ์— ์ ์ ˆํ•œ ์กฐ๊ฑด์—์„œ ๋ฐฐ์–‘ํ•  ๊ฒƒ์„ ๊ถŒ์žฅํ•ฉ๋‹ˆ๋‹ค.

Collect, Washing
ํšจ๋ชจ๋ฅผ 2,000ร—g์—์„œ 5๋ถ„๊ฐ„ ์›์‹ฌ๋ถ„๋ฆฌํ•˜์—ฌ ์ง‘๊ท ํ•ฉ๋‹ˆ๋‹ค. Pellet์— 5~10 mL์˜ ์ฆ๋ฅ˜์ˆ˜๋ฅผ ๋„ฃ์–ด ํ˜„ํƒํ•˜์—ฌ ๋‹ค์‹œ 2,000ร—g์—์„œ 5๋ถ„๊ฐ„ ์›์‹ฌ๋ถ„๋ฆฌํ•˜์—ฌ ์ง‘๊ท ํ•ฉ๋‹ˆ๋‹ค.
* ์„ธ์ฒ™ ํ›„์˜ Pellet์€ -80โ„ƒ์—์„œ ๋™๊ฒฐ๋ณด์กด์ด ๊ฐ€๋Šฅํ•ฉ๋‹ˆ๋‹ค. ์ผ๋ฐ˜์ ์œผ๋กœ ํ•œ ๋ฒˆ ๋™๊ฒฐํ•˜๋ฉด ์ถ”์ถœ ํšจ์œจ์ด ๋†’์•„์ง‘๋‹ˆ๋‹ค.

Pretreatment
์›์‹ฌ๋ถ„๋ฆฌํ•œ ์ƒ๋“ฑ์•ก์„ ์ œ๊ฑฐํ•˜๊ณ , ๊ท ์ฒด๋ฅผ vortexํ•˜์—ฌ ์„ธํฌ๋ฅผ ์ž˜ ํ’€์–ด์ค๋‹ˆ๋‹ค(์ค‘์š”). 0.5 mL์˜ Yest PreLysis buffer๋ฅผ ์ฒจ๊ฐ€ํ•˜์—ฌ vortexํ•œ ํ›„, ์‹ค์˜จ์—์„œ 5๋ถ„๊ฐ„ ์ •์น˜ํ•ฉ๋‹ˆ๋‹ค.
* ์‹คํ—˜์— ์ง€์ •์ด ์—†๋Š” ๊ฒฝ์šฐ์—๋Š”, DTT๋ฅผ 5~50 mM ๋†๋„๋กœ ์ฒจ๊ฐ€ํ•˜๋ฉด ๋‹จ๋ฐฑ์งˆ ์ถ”์ถœ ํšจ์œจ์ด ๋†’์•„์ง‘๋‹ˆ๋‹ค.

๊ท ์ฒด ์šฉํ•ด
์„ธํฌํ˜„ํƒ์•ก์„ 10,000ร—g์—์„œ 5์ดˆ๊ฐ„(4โ„ƒ) ์›์‹ฌ๋ถ„๋ฆฌํ•˜๊ณ  ํšŒ์ˆ˜ํ•œ ๊ท ์ฒด๋ฅผ vortexํ•˜์—ฌ Pellet์„ ์ž˜ ํ’€์–ด์ค๋‹ˆ๋‹ค(์ค‘์š”).
0.5 mL์˜ EzBactYeast Lysis buffer๋ฅผ ์ฒจ๊ฐ€ํ•˜์—ฌ ์‹ค์˜จ์—์„œ 10๋ถ„๊ฐ„ ์ •์น˜ํ•ฉ๋‹ˆ๋‹ค.
* EzBactYeast Lysis buffer ์—๋Š” ๋ฏธ๋ฆฌ WSE-7423 EzBactYeastCrusher์— ์ฒจ๋ถ€๋œ Protease Inihibitor ์™€ DNase I ๋ฅผ ์ฒจ๊ฐ€ํ•ฉ๋‹ˆ๋‹ค.
* ์ถ”์ถœํšจ์œจ์„ ๋†’์ผ ๊ฒฝ์šฐ์—๋Š” ์‚ฐ์„ธ์ฒ™ํ•œ Glass beads (ฯ†0.5 mm)๋ฅผ ์‚ฌ์šฉํ•ฉ๋‹ˆ๋‹ค (Schizosaccharomyces pombe๋Š” ํ•„์ˆ˜).

๋‹จ๋ฐฑ์งˆ Extraction
์„ธํฌ์šฉํ•ด์•ก์„ 4โ„ƒ, 10,000ร—g์—์„œ 5๋ถ„๊ฐ„ ์›์‹ฌ๋ถ„๋ฆฌํ•˜์—ฌ ์ƒ๋“ฑ์•ก์„ ํšŒ์ˆ˜ํ•ฉ๋‹ˆ๋‹ค.

์ผ๋ฐ˜์ ์ธ ํšจ๋ชจ ๋‹จ๋ฐฑ์งˆ ์ถ”์ถœ๋ฐฉ๋ฒ•

1. ํšจ๋ชจ๋ฅผ ํŒŒ์‡„์šฉ ์šฉ์•ก(0.33 M Sucrose, 0.3 M Tris/pH 8.0, 1 mM EDTA, 1 mM EGTA, 2 mM DTT, 100 mM 6-Aminohexanoic acid)์œผ๋กœ Washingํ•œ๋‹ค.
2. ํŒŒ์‡„์šฉ๊ธฐ์— ๊ท ์ฒด๋ฅผ ๋„ฃ๊ณ  ์šฉ๊ธฐ์˜ 40% ์šฉ๋Ÿ‰์˜ ์‚ฐ์„ธ์ •ํ•œ Glass beads๋ฅผ ์ฒจ๊ฐ€ํ•œ๋‹ค.
3. Bead homogenizer์— ์žฅ์ฐฉํ•˜๊ณ  Pulse๋ฅผ 1๋ถ„๋™์•ˆ, ์ด 3~5๋ถ„๊ฐ„ ํŒŒ์‡„ํ•œ๋‹ค.
4. ํŒŒ์‡„ ํ›„, ํ•„์š”ํ•  ๊ฒฝ์šฐ 1 M Tris/pH 7.4๋ฅผ ์ฒจ๊ฐ€ํ•˜์—ฌ pH๋ฅผ ์กฐ์ •ํ•œ๋‹ค.
5. 14,000ร—g์—์„œ 30๋ถ„๊ฐ„ ์›์‹ฌ๋ถ„๋ฆฌํ•˜์—ฌ ์ƒ๋“ฑ์•ก์„ ํšŒ์ˆ˜ํ•œ๋‹ค.

3-2. ์ฐธ๊ณ  ๋ฐ์ดํ„ฐ

์‹คํ—˜์˜ˆ3: ํšจ๋ชจ ๋‹จ๋ฐฑ์งˆ ์ถ”์ถœ ํšจ์œจ์˜ ๋น„๊ต

์ถ”์ถœ์— ์‚ฌ์šฉํ•œ ์‹œ์•ฝ

P: PBS
A:ย WSE-7423 EzBactYeastCrusher
C: ํƒ€์‚ฌ ์‹œํŒ ์‹œ์•ฝ
Y: ํƒ€์‚ฌ ์‹œํŒ ์‹œ์•ฝ
AD:ย WSE-7423 EzBactYeastCrusherย + 5 mM DTT

์ด ์‚ฌ์ง„์€ YPD ๋ฐฐ์ง€์—์„œ ๋ฐฐ์–‘ํ•œย Saccharomyces cerevisiae 5 mL์˜ ๋ฐฐ์–‘์•ก์—์„œ ์„ธํฌ๋ฅผ ํšŒ์ˆ˜ํ•˜๊ณ , ์œ„์— ๊ธฐ์žฌํ•œ ๋ฐฉ๋ฒ•์œผ๋กœ ๋‹จ๋ฐฑ์งˆ์„ ์ถ”์ถœํ•˜์—ฌ ์ „๊ธฐ์˜๋™ํ•œ ๊ฒฐ๊ณผ์ž…๋‹ˆ๋‹ค. Glass beads๋ฒ•์€ ์„ธํฌ์˜ ์•ฝ 2.5๋ฐฐ ์šฉ๋Ÿ‰์˜ Glass beads์™€ ๊ฐ ์ถ”์ถœ์•ก 0.2 mL์„ ์ฒจ๊ฐ€ํ•˜๊ณ  10๋ถ„๊ฐ„ vortexํ•˜์—ฌ ๋‹จ๋ฐฑ์งˆ์„ ์ถ”์ถœํ•˜์˜€์Šต๋‹ˆ๋‹ค. Zymolyase์ฒ˜๋ฆฌ๋ฒ•์€ 5 mg/mL์˜ Zymolyase๋ฅผ 37โ„ƒ์—์„œ 30๋ถ„๊ฐ„ ์ฒ˜๋ฆฌํ•œ ํ›„, ์„ธํฌ๋ฅผ PBS๋กœ Washingํ•˜๊ณ  ์„ธํฌ์— ๊ฐ ์ถ”์ถœ์•ก์„ 0.2 mL ์ฒจ๊ฐ€ํ•˜์—ฌ 10๋ถ„๊ฐ„ Incubationํ•˜์—ฌ ๋‹จ๋ฐฑ์งˆ์„ ์ถ”์ถœํ•˜์˜€์Šต๋‹ˆ๋‹ค. ์ „์ฒ˜๋ฆฌ ์—†์Œ๊ณผ Pretreatment๋Š” ์„ธํฌ์— ๊ฐ ์ถ”์ถœ์•ก์„ 0.2 mL ์ฒจ๊ฐ€ํ•˜์—ฌ 10๋ถ„๊ฐ„ Incubationํ•˜์—ฌ ๋‹จ๋ฐฑ์งˆ์„ ์ถ”์ถœํ•˜์˜€์Šต๋‹ˆ๋‹ค. Pretreatment๋Š” ๋‹จ๋ฐฑ์งˆ ์ถ”์ถœ ์ „์—ย WSE-7423 EzBactYeastCrusher์— ํฌํ•จ๋œ Pre-Lysis buffer๋ฅผ ์„ธํฌ์— ์ฒจ๊ฐ€ํ•˜์—ฌ 10๋ถ„๊ฐ„ Incubationํ•˜์˜€์Šต๋‹ˆ๋‹ค. Glass beads๋ฒ•, Zymolyase๋ฒ•์„ ํฌํ•จํ•˜์—ฌย WSE-7423 EzBactYeastCrusher์—์„œ ์ถ”์ถœํ•œ lane(A)์˜ ๋‹จ๋ฐฑ์งˆ์€ ๊ธฐํƒ€ ์ถ”์ถœ์‹œ์•ฝ์— ๋น„ํ•ด ๋‹จ๋ฐฑ์งˆ ๋ฐด๋“œ๊ฐ€ ๋งŽ๊ณ , ๋†๋„๋„ ๋†’์€ ๊ฒƒ์œผ๋กœ ๋‚˜ํƒ€๋‚ฌ์Šต๋‹ˆ๋‹ค. ํŠนํžˆ, ์šฉ์ถœ์ด ์–ด๋ ค์šด ๊ณ ๋ถ„์ž ์˜์—ญ์˜ ๋‹จ๋ฐฑ์งˆ ์ถ”์ถœ ํšจ์œจ์ด ๋†’๊ณ , ๋‹ค์–‘ํ•œ ๋‹จ๋ฐฑ์งˆ์ด ๊ณ ํšจ์œจ๋กœ ์šฉ์ถœ๋  ์ˆ˜ ์žˆ์Œ์„ ์•Œ ์ˆ˜ ์žˆ์Šต๋‹ˆ๋‹ค.

์‹คํ—˜์˜ˆ4: ํšจ๋ชจ ์ถ”์ถœ ๋‹จ๋ฐฑ์งˆ์˜ Immunoprecipitation (๋ฉด์—ญ์นจ๊ฐ•)

A: Non-induced ํšจ๋ชจ์ถ”์ถœ๋‹จ๋ฐฑ์งˆ๋กœ IP
B: Induced ํšจ๋ชจ์ถ”์ถœ๋‹จ๋ฐฑ์งˆ (with DTT)๋กœ IP
C. Induced ํšจ๋ชจ์ถ”์ถœ๋‹จ๋ฐฑ์งˆ (without DTT)๋กœ IP

๊ทธ๋ฆผ์€ V5 tag-EGFP ๋ฐœํ˜„ ํšจ๋ชจ์—์„œ WSE-7423 EzBactYeastCrusher๋ฅผ ์‚ฌ์šฉํ•˜์—ฌ ์ถ”์ถœํ•œ ๋‹จ๋ฐฑ์งˆ์„ V5 ํ•ญ์ฒด๋ฅผ ์‚ฌ์šฉํ•˜์—ฌ ๋ฉด์—ญ ์นจ๊ฐ•ํ•œ ๊ฒฐ๊ณผ๋ฅผ ๋‚˜ํƒ€๋ƒ…๋‹ˆ๋‹ค. ์•„๋ž˜ ์‚ฌ์ง„์€ ์ถ”์ถœ ํ›„ ๋‹จ๋ฐฑ์งˆ์„ Blue LEDํ•˜์—์„œ EGFP์˜ ํ˜•๊ด‘์„ ๊ฒ€์ถœํ•œ ์ด๋ฏธ์ง€์ž…๋‹ˆ๋‹ค. ์ „๊ธฐ์˜๋™ ํŒจํ„ด์˜ ๋ ˆ์ธA๋Š” ๋ฐœํ˜„ ์œ ๋„ํ•˜์ง€ ์•Š์•˜๊ธฐ ๋•Œ๋ฌธ์— EGFP ๋ฐด๋“œ๋Š” ๊ฒ€์ถœ๋˜์ง€ ์•Š์•˜์Šต๋‹ˆ๋‹ค. ๊ทธ๋Ÿฌ๋‚˜ ๋ฐœํ˜„ ์œ ๋„ํ•œ ๋ ˆ์ธ B์™€ C์—๋Š” ๋ช…๋ฃŒํ•˜๊ฒŒ EGFP ๋ฐด๋“œ๊ฐ€ ๊ฒ€์ถœ๋˜์—ˆ์Šต๋‹ˆ๋‹ค. ๋ ˆ์ธ B๋Š” ํ™˜์›์ œ์ธ DTT๋ฅผ ์ฒจ๊ฐ€ํ•œ ์ƒํƒœ์—์„œ ๋‹จ๋ฐฑ์งˆ์„ ์ถ”์ถœํ•˜์˜€๊ธฐ ๋•Œ๋ฌธ์— ๋ฉด์—ญ์นจ๊ฐ•์—์„œ ์‚ฌ์šฉํ•œ ํ•ญ์ฒด์œ ๋ž˜ ๋ฐด๋“œ๋„ ํ™˜์›๋˜์–ด Heavy chain(75kDa)๊ณผ Light chain(25kDa)์œผ๋กœ ๋‚˜๋‰ฉ๋‹ˆ๋‹ค. ํ•œํŽธ ๋ ˆ์ธ A์™€ C๋Š” ํ™˜์›์ œ๋ฅผ ์ฒจ๊ฐ€ํ•˜์ง€ ์•Š๊ณ  ์ถ”์ถœํ•˜์—ฌ ํ•ญ์ฒด๊ฐ€ ํ™˜์›๋˜์ง€ ์•Š๊ณ  150kDa ๋ฐด๋“œ๋กœ ๊ฒ€์ถœ๋˜์—ˆ์Šต๋‹ˆ๋‹ค. ์ด์™€ ๊ฐ™์ด WSE-7423 EzBactYeastCrusher๋ฅผ ์‚ฌ์šฉํ•˜์—ฌ ์ถ”์ถœํ•œ ๋‹จ๋ฐฑ์งˆ์€ ๋‹จ๋ฐฑ์งˆ์˜ ๋ณ€์„ฑ์ด ์ตœ์†Œํ•œ์œผ๋กœ ์–ต์ œ๋˜์–ด ์žˆ์–ด Antigen site-Antibody complex๊ฐ€ ์†์ƒ๋˜์ง€ ์•Š๊ณ  ํ™œ์„ฑ์„ ์œ ์ง€ํ•œ ์ƒํƒœ(EGFP์˜ ํ˜•๊ด‘)์ž„์„ ์•Œ ์ˆ˜ ์žˆ์Šต๋‹ˆ๋‹ค.

ย  4. ๋™๋ฌผ์„ธํฌ์˜ ๋‹จ๋ฐฑ์งˆ ์ถ”์ถœ ๋ฐฉ๋ฒ•

๋น„๊ต์  ๋ถ€๋“œ๋Ÿฌ์šด ์กฐ์ง์˜ ๊ฒฝ์šฐ Potterํ˜• Homogenizer๊ฐ€, ๋˜ํ•œ ๋Œ€๋Ÿ‰์˜ ์กฐ์ง์„ ๋‹ค๋ฃจ๋Š” ๊ฒฝ์šฐ Waring Blender๋‚˜ Blender๊ฐ€ ์ด์šฉ๋˜๊ณ  ์žˆ์Šต๋‹ˆ๋‹ค. ๋ฌผ๋ก  ๋ฏธ์ƒ๋ฌผ๊ณผ ๋งˆ์ฐฌ๊ฐ€์ง€๋กœ ์ดˆ์ŒํŒŒ ํŒŒ์‡„๋‚˜ ๋™๊ฒฐ ์œตํ•ด๋ฒ•์— ์˜ํ•œ ํŒŒ์‡„๋„ ์ด์šฉ๋ฉ๋‹ˆ๋‹ค. ๋˜ ๋™๋ฌผ ์„ธํฌ์—๋Š” ์„ธํฌ๋ฒฝ์ด ์—†๊ธฐ ๋•Œ๋ฌธ์—, ์‚ผํˆฌ์••์— ์˜ํ•ด ์„ธํฌ๋ง‰์„ ํŒŒ๊ดดํ•ด ์†Œ๊ธฐ๊ด€ ๋“ฑ์„ ๋ถ„ํšํ•˜๋Š” ๋ฐฉ๋ฒ•์ด ์žˆ์Šต๋‹ˆ๋‹ค. ์–ด๋–ค ์ข…๋ฅ˜์˜ ํšจ์†Œ ์ถ”์ถœ์—๋Š” ์œ ๊ธฐ์šฉ๋งค(์•„์„ธํ†ค)์— ์˜ํ•œ ์ถ”์ถœ๋ฒ•์ด ์ด์šฉ๋ฉ๋‹ˆ๋‹ค. ๊ทธ ์™ธ ํšจ์†Œ๋‚˜ ๊ณ„๋ฉดํ™œ์„ฑ์ œ๋ฅผ ์ด์šฉํ•œ ์ถ”์ถœ๋ฐฉ๋ฒ•๋„ ์ผ๋ฐ˜์ ์œผ๋กœ ์ž์ฃผ ์ด์šฉ๋ฉ๋‹ˆ๋‹ค. ATTO์˜ WSE-7420 EzRIPA Lysis kit๋Š” Protease Inhibitor์™€ Phosphatase Inhibitor๊ฐ€ ํฌํ•จ๋˜์–ด ์žˆ์–ด ์„ธํฌ์— ์„ž๋Š” ๊ฒƒ๋งŒ์œผ๋กœ ๋‹จ๋ฐฑ์งˆ์„ ์•ˆ์ •์ ์œผ๋กœ ์ถ”์ถœํ•  ์ˆ˜ ์žˆ์Šต๋‹ˆ๋‹ค.

4-1. ์‹คํ—˜๋ฐฉ๋ฒ•

์‹คํ—˜์žฌ๋ฃŒ
๋ฐฐ์–‘์„ธํฌ (10 cm dish, 5~20ร—10โถ cells)
WSE-7420 EzRIPA Lysis kit
WSE-7430 EzPBS
๋งˆ์ดํฌ๋กœ ์›์‹ฌํŠœ๋ธŒ, ํ”ผํŽซ tip ๋“ฑ
์›์‹ฌ๊ธฐ, ๋งˆ์ดํฌ๋กœํ”ผํŽซ ๋“ฑ

Cell culture
์„ธํฌ๋ฅผ ์ ๋‹นํ•œ ๋ฐฐ์ง€์—์„œ Confluentํ• ๋•Œ๊นŒ์ง€ ๋ฐฐ์–‘ํ•ฉ๋‹ˆ๋‹ค. (5~20ร—10โถ cells ์ƒ๋‹น)
* ์‚ฌ์šฉํ•˜๋Š” ์„ธํฌ์— ๋”ฐ๋ผ ์„ธํฌ์˜ ํฌ๊ธฐ๋‚˜ ๋ฐ€๋„์— ๋”ฐ๋ผ ํšŒ์ˆ˜๋˜๋Š” ์„ธํฌ ์ˆ˜๋Š” ๋‹ค๋ฆ…๋‹ˆ๋‹ค.

Washing
WSE-7430 EzPBS๋กœ 2ํšŒ ์„ธ์ฒ™ํ•ฉ๋‹ˆ๋‹ค.
* WSE-7430 EzPBS๋Š” ์‚ฌ์šฉ์ „์— ์ฆ๋ฅ˜์ˆ˜๋กœ 10๋ฐฐ ํฌ์„ํ•ฉ๋‹ˆ๋‹ค.
* Adherent cell์€ ๋–ผ์–ด๋‚ด์ง€ ์•Š๊ณ  Washingํ•ฉ๋‹ˆ๋‹ค. Suspension cell์€ Microcentrifuge tube์— ์ง‘๊ท ํ•˜์—ฌ WSE-7430 EzPBS์— ํ˜„ํƒํ•˜์—ฌ Washingํ•ฉ๋‹ˆ๋‹ค.
* ์„ธ์ • ํ›„ ์„ธํฌ๋Š” -80โ„ƒ์— ๋ณด๊ด€ ๊ฐ€๋Šฅํ•ฉ๋‹ˆ๋‹ค.

์„ธํฌ ์šฉํ•ด
1.0 mL์˜ RIPA Lysis buffer๋ฅผ ์ฒจ๊ฐ€ํ•˜์—ฌ ice์— 15๋ถ„๊ฐ„ ์ •์น˜ํ•ฉ๋‹ˆ๋‹ค.
* RIPA Lysis buffer์—๋Š” ๋ฏธ๋ฆฌย WSE-7420 EzRIPA Lysis kit์— ํฌํ•จ๋œ Protease Inhibitor์™€ Phosphatase Inhibitor๋ฅผ ์ฒจ๊ฐ€ํ•ฉ๋‹ˆ๋‹ค.
* Suspension cell์€ ์›์‹ฌ๋ถ„๋ฆฌ ์‹œ RIPA Lysis buffer (์กฐ์ • ์™„๋ฃŒ)๋ฅผ ์ฒจ๊ฐ€ํ•˜์—ฌ Pipetting์œผ๋กœ ํ’€์–ด์ค€ ํ›„, ice์— 15๋ถ„๊ฐ„ ์ •์น˜ํ•ฉ๋‹ˆ๋‹ค.

์ถ”์ถœ์•ก ํšŒ์ˆ˜
์„ธํฌ๋ฅผ Scraper ๋“ฑ์œผ๋กœ ๊ธ์–ด๋‚ด์–ด Microcentrifuge tube์— ํšŒ์ˆ˜ํ•ฉ๋‹ˆ๋‹ค. ์ด ์„ธํฌ ์ถ”์ถœ์•ก์„ 15,000ร—g
์„ธํฌ๋ฅผ ์Šคํฌ๋ ˆ์ดํผ ๋“ฑ์œผ๋กœ ๊ธ์–ด๋‚ด์–ด ๋งˆ์ดํฌ๋กœ ์›์‹ฌ ํŠœ๋ธŒ๋กœ ํšŒ์ˆ˜ํ•ฉ๋‹ˆ๋‹ค.์ด ์„ธํฌ ์ถ”์ถœ์•ก์„ 4โ„ƒ, 15,000xg์—์„œ 5~15๋ถ„๊ฐ„ ์›์‹ฌ๋ถ„๋ฆฌํ•ฉ๋‹ˆ๋‹ค.
* Suspension cell๋„ ์œ„์™€ ๋งˆ์ฐฌ๊ฐ€์ง€๋กœ ์›์‹ฌ๋ถ„๋ฆฌํ•ฉ๋‹ˆ๋‹ค.

๋‹จ๋ฐฑ์งˆ ํšŒ์ˆ˜
์ƒ๋“ฑ์•ก์„ ์ƒˆ๋กœ์šด Microcentrifuge tube๋กœ ๋ชจ์•„์ค๋‹ˆ๋‹ค. ๊ฐ€์šฉํ™”๋œ ๋‹จ๋ฐฑ์งˆ ์šฉ์•ก์€ ์‚ฌ์šฉ์‹œ๊นŒ์ง€ ice์—์„œ ๋ณด๊ด€ ๋˜๋Š” -80โ„ƒ์—์„œ ๋ณด๊ด€ํ•ฉ๋‹ˆ๋‹ค.
* ํ•ต๋‹จ๋ฐฑ์งˆ๋„ ์ถ”์ถœํ•˜๋Š” ๊ฒฝ์šฐ๋Š” SDS๋ฅผ ์ตœ์ข…๋†๋„๊ฐ€ 0.5%๊ฐ€ ๋˜๋„๋ก ์ฒจ๊ฐ€ํ•˜๋ฉด ์ถ”์ถœํšจ์œจ์ด ๋†’์•„์ง‘๋‹ˆ๋‹ค. DNA ์šฉ์ถœ๋กœ ์ธํ•ด ์ ์„ฑ์ด ๋†’์•„์ง€๋ฏ€๋กœ ์ดˆ์ŒํŒŒ ํŒŒ์‡„ ๋˜๋Š” DNase I๋ฅผ ์ฒ˜๋ฆฌํ•ฉ๋‹ˆ๋‹ค.

์ผ๋ฐ˜์ ์ธ ๋™๋ฌผ์„ธํฌ ๋‹จ๋ฐฑ์งˆ ์ถ”์ถœ๋ฐฉ๋ฒ•

๋™๋ฌผ์กฐ์งยท์„ธํฌ๋กœ๋ถ€ํ„ฐ ๋‹จ๋ฐฑ์งˆ์„ ์ถ”์ถœํ•˜๊ธฐ ์œ„ํ•ด์„œ๋Š” ์„ธํฌ๋ฅผ ํŒŒ์‡„ํ•ด์•ผ ํ•ฉ๋‹ˆ๋‹ค. ์ผ๋ฐ˜์ ์œผ๋กœ ์ƒ˜ํ”Œ์„ ํŒŒ๊ดดํ•˜๋Š” ๋ฐฉ๋ฒ•์œผ๋กœ๋Š” โ‘  ๋ฌผ๋ฆฌ์ ํŒŒ์‡„ (์šฉ๋งค๋ฅผ ์ƒ˜ํ”Œ์˜ 3~10๋ฐฐ ์ฒจ๊ฐ€ํ•˜์—ฌ ๊ท ์งˆํ•œ๋‹ค), โ‘ก ์ดˆ์ŒํŒŒํŒŒ์‡„(50 mg/mL ์ดํ•˜์˜ ๋†๋„๋กœ ๋ƒ‰๊ฐํ•˜๋ฉด์„œ 1~10๋ถ„๊ฐ„ ์ดˆ์ŒํŒŒ๋ฅผ ๊ฐ€ํ•œ๋‹ค), โ‘ข ๋™๊ฒฐ์œตํ•ด, โ‘ฃ ๊ฐ€์••ํŒŒ์‡„ (French press ๋“ฑ), โ‘ค ์‚ผํˆฌ์••์‡ผํฌ (pH, ์ด์˜จ๊ฐ•๋„ ๋ณ€ํ™”), โ‘ฅ ์œ ๊ธฐ์šฉ๋งค (์•„์„ธํ†ค, ์•Œ์ฝ”์˜ฌ์— ์˜ํ•ด ์ง€์งˆ๋ง‰ ๊ตฌ์กฐ ํŒŒ๊ดด), โ‘ฆ ํšจ์†Œ์ฒ˜๋ฆฌ (์„ธํฌ๋ฒฝ์˜ ํšจ์†Œ ํŒŒ๊ดด ๋“ฑ), โ‘ง ๊ณ„๋ฉดํ™œ์„ฑ์ œ ์ด์šฉ ๋“ฑ์„ ๋“ค ์ˆ˜ ์žˆ์Šต๋‹ˆ๋‹ค.

ย  5. ์„ธํฌ์†Œ๊ธฐ๊ด€์˜ ๋‹จ๋ฐฑ์งˆ ์ถ”์ถœ ๋ฐฉ๋ฒ•

์„ธํฌ ๋‚ด์˜ ๋‹จ๋ฐฑ์งˆ์€ ์„ธํฌ์งˆ๊ณผ ํ•ต, ๋ฏธํ† ์ฝ˜๋“œ๋ฆฌ์•„ ๋“ฑ ์–ด๋–ค ํŠน์ • ์˜์—ญ์— ๋ถ„ํฌํ•˜๋Š” ๋‹จ๋ฐฑ์งˆ์ด ์žˆ์Šต๋‹ˆ๋‹ค. ์ด ๋‹จ๋ฐฑ์งˆ์€ ์ž๊ทน์ด๋‚˜ ์„ธํฌ์ฃผ๊ธฐ ๋“ฑ์˜ ์˜ํ–ฅ์„ ๋ฐ›์•„ ์œ„์น˜๋‚˜ ๊ธฐ๋Šฅ์ด ๋ณ€ํ™”ํ•˜๋Š” ๊ฒฝ์šฐ๊ฐ€ ์žˆ์Šต๋‹ˆ๋‹ค. ์„ธํฌ ์ „์ฒด์—๋Š” ๋งค์šฐ ๋งŽ์€ ๋‹จ๋ฐฑ์งˆ์ด ์กด์žฌํ•˜๋ฏ€๋กœ Target protein์˜ identification์ด๋‚˜ ๋ถ„๋ฆฌ, ์ •์ œ๊ฐ€ ์–ด๋ ค์šด ๊ฒฝ์šฐ๊ฐ€ ์žˆ์Šต๋‹ˆ๋‹ค. ์ด๋Ÿฐ ๊ฒฝ์šฐ ์„ธํฌ๋ฅผ ์„ธํฌ์†Œ๊ธฐ๊ด€๋ณ„๋กœ ๋‚˜๋ˆ„์–ด ํ•ด์„ํ•จ์œผ๋กœ์จ ๋‹จ๋ฐฑ์งˆ์„ ์ถ”์ถœํ•  ์ˆ˜ ์žˆ์–ด Target protein์˜ ๋™์ •์ด๋‚˜ ๊ธฐ๋Šฅ ํ•ด์„์„ ๋ณด๋‹ค ๊ฐ„๋‹จํ•˜๊ฒŒ ํ•  ์ˆ˜ ์žˆ๊ฒŒ ๋ฉ๋‹ˆ๋‹ค.์•„ํ† ์˜ WSE-7421 EzSubcell Extract๋Š” Protease Inhibitor์™€ DNase I์ด ํฌํ•จ๋˜์–ด ์žˆ์–ด ์„ธํฌ์— ์„ž๋Š” ๊ฒƒ๋งŒ์œผ๋กœ ์„ธํฌ์งˆ ๋ถ„ํš, ํ•ต๋ถ„ํš, ๋ฏธํ† ์ฝ˜๋“œ๋ฆฌ์•„๋“ฑ์˜ ๋ง‰๋ถ„ํš, ๋ถˆ์šฉ์„ฑ ๋ถ„ํš ๊ฐ๊ฐ์„ ๋ถ„๋ฆฌยท์ถ”์ถœ ๊ฐ€๋Šฅํ•ฉ๋‹ˆ๋‹ค.

5-1. ์‹คํ—˜๋ฐฉ๋ฒ•

์‹คํ—˜์žฌ๋ฃŒ
๋ฐฐ์–‘์„ธํฌ (10 cm dish, 5~20ร—10โถ cells)
WSE-7421 EzSubcell Extract
WSE-7430 EzPBS
๋งˆ์ดํฌ๋กœ ์›์‹ฌํŠœ๋ธŒ, ํ”ผํŽซ tip ๋“ฑ
์›์‹ฌ๊ธฐ, ๋งˆ์ดํฌ๋กœํ”ผํŽซ ๋“ฑ

Cell culture
์„ธํฌ๋ฅผ ์ ๋‹นํ•œ ๋ฐฐ์ง€์—์„œ Confluentํ• ๋•Œ๊นŒ์ง€ ๋ฐฐ์–‘ํ•ฉ๋‹ˆ๋‹ค. (5~20ร—10โถ cells ์ƒ๋‹น)
* ์‚ฌ์šฉํ•˜๋Š” ๋ฐฐ์ง€์— ๋”ฐ๋ผ ์„ธํฌ์˜ ํฌ๊ธฐ๋‚˜ ๋ฐ€๋„์— ๋”ฐ๋ผ ํšŒ์ˆ˜๋˜๋Š” ์„ธํฌ ์ˆ˜๋Š” ๋‹ค๋ฆ…๋‹ˆ๋‹ค.

์„ธํฌ ํšŒ์ˆ˜
์„ธํฌ๋ฅผ WSE-7430 EzPBS๋กœ Washingํ•˜๊ณ  Trypsin ๋“ฑ์œผ๋กœ Detachํ•˜์—ฌ ํšŒ์ˆ˜ํ•ฉ๋‹ˆ๋‹ค. ํšŒ์ˆ˜ ํ›„ ์„ธํฌ๋Š” ๋‹ค์‹œ WSE-7430 EzPBS๋กœ Washingํ•ฉ๋‹ˆ๋‹ค.
* ๋ฌผ๋ฆฌ์  ํŒŒ๊ดด๋ฅผ ํ”ผํ•˜๊ธฐ ์œ„ํ•ด Scraper ๋“ฑ์œผ๋กœ ๋–ผ์–ด๋‚ด์ง€ ์•Š๊ณ  ์„ธํฌ๋Š” Dissociationํ•˜์—ฌ ํšŒ์ˆ˜ํ•ฉ๋‹ˆ๋‹ค.
* WSE-7430 EzPBS๋Š” ์‚ฌ์šฉ ์ „์— ์ฆ๋ฅ˜์ˆ˜๋กœ 10๋ฐฐ ํฌ์„ํ•ฉ๋‹ˆ๋‹ค.
* Suspension cell์€ Microcentrifuge tube์— ์ง‘๊ท ํ•˜์—ฌ WSE-7430 EzPBS์— ํ˜„ํƒํ•˜์—ฌ Washingํ•ฉ๋‹ˆ๋‹ค. Washing ํ›„ ์„ธํฌ๋Š” -80โ„ƒ์—์„œ ๋ณด๊ด€ ๊ฐ€๋Šฅํ•ฉ๋‹ˆ๋‹ค. ๋™๊ฒฐ ํ›„ ์„ธํฌ์—์„œ๋„ ์ถ”์ถœ ๊ฐ€๋Šฅํ•ฉ๋‹ˆ๋‹ค.

์„ธํฌ์งˆ ์ถ”์ถœ
์„ธํฌ์— ice์—์„œ ์ฐจ๊ฐ‘๊ฒŒ ํ•œ 1 mL์˜ Extraction buffer 1์„ ์ฒจ๊ฐ€ํ•œ ํ›„, vortex๋กœ ํ˜ผํ•ฉํ•˜์—ฌ 4โ„ƒ์—์„œ 10๋ถ„๊ฐ„ incubationํ•ฉ๋‹ˆ๋‹ค. 4โ„ƒ, 700ร—g์—์„œ 5๋ถ„๊ฐ„ ์›์‹ฌ๋ถ„๋ฆฌํ•˜์—ฌ ์ƒ๋“ฑ์•ก์„ Microcentrifuge tube์— ํšŒ์ˆ˜ํ•ฉ๋‹ˆ๋‹ค. (์„ธํฌ์งˆ ๋ถ„ํš)

์„ธํฌ๋ง‰ ์ถ”์ถœ
Pellet์— ice์—์„œ ์ฐจ๊ฐ‘๊ฒŒ ํ•œ 1 mL์˜ Extraction buffer 2๋ฅผ ์ฒจ๊ฐ€ํ•œ ํ›„, vortex๋กœ ํ˜ผํ•ฉํ•˜์—ฌย 4โ„ƒ์—์„œ 30๋ถ„๊ฐ„ incubationํ•ฉ๋‹ˆ๋‹ค.ย 4โ„ƒ, 4,000ร—g์—์„œ 5๋ถ„๊ฐ„ ์›์‹ฌ๋ถ„๋ฆฌํ•˜์—ฌ ์ƒ๋“ฑ์•ก์„ Microcentrifuge tube์— ํšŒ์ˆ˜ํ•ฉ๋‹ˆ๋‹ค. (์„ธํฌ๋ง‰ ๋ถ„ํš)
* ์„ธํฌ๋ง‰ ๋ถ„ํš์—๋Š” ๋ฏธํ† ์ฝ˜๋“œ๋ฆฌ์•„, ๊ณจ์ง€์ฒด, ์†Œํฌ์ฒด ๋“ฑ์ด ํฌํ•จ๋ฉ๋‹ˆ๋‹ค.

์„ธํฌํ•ต ์ถ”์ถœ
Pellet์— ice์— ์ฐจ๊ฐ‘๊ฒŒ ํ•œ 0.5 mL์˜ Extraction buffer 3์„ ์ฒจ๊ฐ€ํ•œ ํ›„, vortex๋กœ ํ˜ผํ•ฉํ•˜์—ฌย 4โ„ƒ์—์„œ 60๋ถ„๊ฐ„ incubationํ•ฉ๋‹ˆ๋‹ค.ย 4โ„ƒ, 9,000ร—g์—์„œ 5๋ถ„๊ฐ„ ์›์‹ฌ๋ถ„๋ฆฌํ•˜์—ฌ ์ƒ๋“ฑ์•ก์„ Microcentrifuge tube์— ํšŒ์ˆ˜ํ•ฉ๋‹ˆ๋‹ค. (ํ•ต ๋ถ„ํš)

Insoluble ์ถ”์ถœ
Pellet์— ์ƒ์˜จ์˜ 0.5 mL์˜ Extraction buffer 4๋ฅผ ์ฒจ๊ฐ€ํ•˜์—ฌ vortexํ•˜์—ฌ ์šฉํ•ดํ•ฉ๋‹ˆ๋‹ค.

5-2. ์ฐธ๊ณ  ๋ฐ์ดํ„ฐ

์ธ๊ฐ„ ๊ฒฐ์žฅ์•” ์œ ๋ž˜ ์„ธํฌ SW480(2ร—10โท cells)๋ฅผ WSE-7420 EzRIPA Lysis kit ๋ฐ WSE-7421 EzSubcell Extract๋ฅผ ์‚ฌ์šฉํ•˜์—ฌ ์ „ ์„ธํฌ (Whole cell), ์„ธํฌ์งˆ ๋ถ„ํš(Cytosol, Ext.1), ์„ธํฌ๋ง‰ ๋ถ„ํš(Membrane, Ext.2), ์„ธํฌํ•ต ๋ถ„ํš(Nuclei, Ext.3), Insoluble ๋ถ„ํš(Insoluble, Ext.4)์˜ ๋‹จ๋ฐฑ์งˆ์„ ์ถ”์ถœํ•˜์—ฌ AE-1430 EzApply๋ฅผ ์ด์šฉํ•˜์—ฌ ์ „๊ธฐ์˜๋™ ์ƒ˜ํ”Œ์„ ์ค€๋น„ํ•˜์˜€์Šต๋‹ˆ๋‹ค. e-PAGEL (5-20% Gradient gel)๋กœ ๋ถ„๋ฆฌํ•˜๊ณ  AE-1465 EzFastBlot์„ ์‚ฌ์šฉํ•˜์—ฌ ClearBlot P plus Membrane์— ์ „์‚ฌํ•˜์—ฌ AE-1477 EzBlock CAS๋กœ 30๋ถ„๊ฐ„ Blockingํ•˜์˜€์Šต๋‹ˆ๋‹ค. Blocking ํ›„ Membrane์„ Mitochondria ๋งˆ์ปค์˜ Cytochrome Cํ•ญ์ฒด, ์ด์–ด์„œ HRPํ‘œ์ง€ 2์ฐจํ•ญ์ฒด์™€ ๋ฐ˜์‘ํ•˜์—ฌ WSE-7120 EzWestLumi plus์—์„œ ๊ฒ€์ถœํ•˜์˜€์Šต๋‹ˆ๋‹ค. (๊ทธ๋ฆผ์˜ ์ฒซ๋ฒˆ์งธ ์ด๋ฏธ์ง€) Whole cell ์ถ”์ถœ์•ก๊ณผ ์„ธํฌ๋ง‰ ๋ถ„ํš์—์„œ๋งŒ ๋ฐด๋“œ๊ฐ€ ๊ฒ€์ถœ๋˜์–ด, Mitochondria๋ฅผ ํฌํ•จํ•œ ์„ธํฌ๋ง‰ ๋ถ„ํš์ด ๋‹ค๋ฅธ ์†Œ๊ธฐ๊ด€์˜ ์˜ค์—ผ์—†์ด ๋ถ„๋ฆฌ๋œ ๊ฒƒ์ด ๋‚˜ํƒ€๋‚ฌ์Šต๋‹ˆ๋‹ค.
์ด Membrane์„ WSE-7240 EzReprobe๋กœ ํ•ญ์ฒด๋ฅผ Strippingํ•œ ํ›„, ๋‹ค์‹œย AE-1477 EzBlock CAS๋กœ Blocking ํ›„, ์„ธํฌํ•ต ๋งˆ์ปค์˜ Lamin A/C ํ•ญ์ฒด, ์ด์–ด์„œ HRPํ‘œ์ง€ 2์ฐจํ•ญ์ฒด์™€ ๋ฐ˜์‘ํ•˜์—ฌย WSE-7120 EzWestLumi plus๋กœ ๊ฒ€์ถœํ•˜์˜€์Šต๋‹ˆ๋‹ค. (๊ทธ๋ฆผ์˜ ๋‘๋ฒˆ์งธ ์ด๋ฏธ์ง€) Whole cell๊ณผ ์„ธํฌํ•ต ๋ถ„ํš์—์„œ๋งŒ ๋ฐด๋“œ๊ฐ€ ๊ฒ€์ถœ๋˜์–ด ์„ธํฌํ•ต ๋ถ„ํš ๋˜ํ•œ ๋‹ค๋ฅธ ์†Œ๊ธฐ๊ด€์˜ ์˜ค์—ผ์—†์ด ๋ถ„๋ฆฌ๋œ ๊ฒƒ์„ ์•Œ ์ˆ˜ ์žˆ์Šต๋‹ˆ๋‹ค.
๋‹ค์‹œ ๋™์ผํ•˜๊ฒŒ Stripping, Blocking์„ ํ•œ ํ›„,ย ฮฒ-Actinํ•ญ์ฒด์™€ ๋ฐ˜์‘ํ•˜์—ฌ ๊ฒ€์ถœํ•˜์˜€์Šต๋‹ˆ๋‹ค. (๊ทธ๋ฆผ์˜ ์„ธ๋ฒˆ์งธ ์ด๋ฏธ์ง€) ์ด Membrane์„ Stripping, Blockingํ•œ ํ›„, GAPDHํ•ญ์ฒด์™€ ๋ฐ˜์‘ํ•˜์—ฌ ๊ฒ€์ถœํ•˜์˜€์Šต๋‹ˆ๋‹ค. (๊ทธ๋ฆผ์˜ ๋„ค๋ฒˆ์งธ ์ด๋ฏธ์ง€)
์„ธํฌ์งˆ ๋งˆ์ปค์ธย ฮฒ-Actin๊ณผ GAPDH๋Š” Whole cell์ถ”์ถœ์•ก๊ณผ ์„ธํฌ์งˆ ๋ถ„ํš์— ๋ฐด๋“œ๊ฐ€ ๊ฒ€์ถœ๋˜์—ˆ์ง€๋งŒ, ์„ธํฌ๋ง‰ ๋ถ„ํš์—๋„ ๊ฒ€์ถœ๋˜๊ณ  ์žˆ์–ด์„œ ์„ธํฌ์งˆ ๋ถ„ํš์ด ๋ง‰ ๋ถ„ํš์— ์•ฝ๊ฐ„ ํ˜ผ์žฌํ•˜๊ณ  ์žˆ๋Š” ๊ฒƒ์ด ๋‚˜ํƒ€๋‚ฌ์Šต๋‹ˆ๋‹ค. ์ด๋Ÿฌํ•œ ๊ฒฝ์šฐ,ย WSE-7421 EzSubcell Extract์— ์˜ํ•œ ์†Œ๊ธฐ๊ด€ ๋ถ„ํš์˜ ์ถ”์ถœ๋ฒ•์€ ์ถ”์ถœ์‹œ๋งˆ๋‹ค Washing์กฐ์ž‘์„ ์‹ค์‹œํ•˜์—ฌ ์˜ค์—ผ์„ ์ค„์ด๊ณ  ์†Œ๊ธฐ๊ด€ ๋ถ„ํš์„ ์ถ”์ถœํ•˜๋Š” ๊ฒƒ์ด ๊ฐ€๋Šฅํ•ฉ๋‹ˆ๋‹ค. ์ด์™€ ๊ฐ™์ดย WSE-7421 EzSubcell Extract๋Š” ๊ฐ ์†Œ๊ธฐ๊ด€ ๋ถ„ํš์„ ์„ธํฌ์™€ ์„ž๋Š” ๊ฒƒ๋งŒ์œผ๋กœ๋„ ์ถ”์ถœ์ด ๊ฐ€๋Šฅํ•˜์—ฌย WSE-7240 EzReprobe์™€ ํ•จ๊ป˜ ์‚ฌ์šฉํ•˜๋ฉด ์ ์€ ์–‘์˜ ์ƒ˜ํ”Œ๋กœ ๋งŽ์€ ๋‹จ๋ฐฑ์งˆ์˜ ๋ฐœํ˜„์„ ์•Œ ์ˆ˜ ์žˆ์Šต๋‹ˆ๋‹ค.

์‹คํ—˜์˜ˆ6: ์ถ”์ถœ์•ก์˜ ๋‹จ๋ฐฑ์งˆ๋Ÿ‰๊ณผ ํ•ต ์—ผ์ƒ‰ ์ด๋ฏธ์ง€

Confluent๋œ 1๊ฐœ์˜ culture dish (ฯ†100 mm, 1~2ร—10โทcells)๋ฅผย WSE-7421 EzSubcell Extract๋กœ ๊ฐ ์„ธํฌ์˜ ์†Œ๊ธฐ๊ด€ ์ถ”์ถœ์•ก์„ ์กฐ์ œํ•˜์˜€์Šต๋‹ˆ๋‹ค. Whole cell์˜ ์ถ”์ถœ์•ก์€ WSE-7420 EzRIPA Lysis kit๋กœ ์กฐ์ œํ•˜์˜€์Šต๋‹ˆ๋‹ค. ๋‹จ๋ฐฑ์งˆ ๋†๋„๋Š” BCA๋ฒ•์„ ์ด์šฉํ•˜์˜€์Šต๋‹ˆ๋‹ค.

WSE-7421 EzSubcell Extract์˜ Extraction buffer 2๋ฅผ ์ฒ˜๋ฆฌํ•œ ํ›„ Pellet์„ Trypan blue๋กœ ์—ผ์ƒ‰ํ•˜์—ฌ ๊ด€์ฐฐํ•œ ๊ฒƒ์ž…๋‹ˆ๋‹ค. ํ•ต ํ˜•์ƒ์— ์ด์ƒ์ด ์—†์ด, ํ•ต๋ง์ด ์œ ์ง€๋œ ์ƒํƒœ๋กœ ๋ถ„๋ฆฌ๋œ ๊ฒƒ์„ ์•Œ ์ˆ˜ ์žˆ์Šต๋‹ˆ๋‹ค. ๋™๊ฒฐ๋ณด์กดํ•œ ์„ธํฌ์—์„œ๋„ ๋งˆ์ฐฌ๊ฐ€์ง€๋กœ ํ•ต ๋ฐ ๊ฐ ์†Œ๊ธฐ๊ด€์˜ ์ถ”์ถœ์•ก์„ ๋ถ„๋ฆฌํ•  ์ˆ˜ ์žˆ์Šต๋‹ˆ๋‹ค.

ย  Related Products

๋Œ€์žฅ๊ท , ํšจ๋ชจ ๋‹จ๋ฐฑ์งˆ ์ถ”์ถœ Kit
WSE-7423 EzBactYeast Crusher

๋Œ€์žฅ๊ท  ๋ฐ ํšจ๋ชจ์™€ ํ˜ผํ•ฉํ•˜๋Š” ๊ฒƒ๋งŒ์œผ๋กœ ์‰ฝ๊ฒŒ ๋‹จ๋ฐฑ์งˆ์„ ์ถ”์ถœํ•  ์ˆ˜ ์žˆ๋Š” Buffer์ž…๋‹ˆ๋‹ค. ์ถ”์ถœํ•œ ๋‹จ๋ฐฑ์งˆ์€ ์ •์ œ, Immunoprecipitation, ํšจ์†Œํ™œ์„ฑ ์ธก์ •, Western blotting ๋“ฑ์— ์‚ฌ์šฉ์ด ๊ฐ€๋Šฅํ•ฉ๋‹ˆ๋‹ค.
ํ‚คํŠธ ๊ตฌ์„ฑํ’ˆ: Yeast PreLysis buffer 100 mL, BactYeastLysis buffer 100 mL, DNase I 1 mL, Protease Inhibitor 1 mL

 

๋™๋ฌผ์„ธํฌ ๋‹จ๋ฐฑ์งˆ ์ถ”์ถœ Kit
WSE-7420 EzRIPA Lysis kit

๋™๋ฌผ์„ธํฌ์—์„œ ๋‹จ๋ฐฑ์งˆ ์ถ”์ถœ์— ๊ฐ€์žฅ ๋งŽ์ด ์‚ฌ์šฉ๋˜๋Š” RIPA buffer์ž…๋‹ˆ๋‹ค. ์œ„์™€ ๋™์ผํ•˜๊ฒŒ ๋‹ค์–‘ํ•œ ์šฉ๋„๋กœ ์‚ฌ์šฉ์ด ๊ฐ€๋Šฅํ•ฉ๋‹ˆ๋‹ค.
ํ‚คํŠธ ๊ตฌ์„ฑํ’ˆ: EzRIPA Lysis buffer 100 mL, Protease Inhibitor 1 mL, Phoshatase Inhihditor 1 mL

 

๋™๋ฌผ์„ธํฌ ๋‹จ๋ฐฑ์งˆ ์ถ”์ถœ Kit (์„ธํฌ ์†Œ๊ธฐ๊ด€ ๋ถ„๋ฆฌ์šฉ)
WSE-7421 EzSubcell Extract

๋™๋ฌผ์„ธํฌ์™€ ํ˜ผํ•ฉํ•˜๋Š” ๊ฒƒ๋งŒ์œผ๋กœ ๊ฐ ์†Œ๊ธฐ๊ด€์˜ ๋‹จ๋ฐฑ์งˆ (์„ธํฌ์งˆ, ๋ง‰, ํ•ต, Insoluble)์„ ๋ถ„๋ฆฌ, ์ถ”์ถœํ•  ์ˆ˜ ์žˆ๋Š” Kit์ž…๋‹ˆ๋‹ค. ์ถ”์ถœํ•œ ๋‹จ๋ฐฑ์งˆ์€ ์ƒ๊ธฐ์™€ ๋™์ผํ•˜๊ฒŒ ๋‹ค์–‘ํ•œ ์šฉ๋„๋กœ ์‚ฌ์šฉ์ด ๊ฐ€๋Šฅํ•ฉ๋‹ˆ๋‹ค.
ํ‚คํŠธ ๊ตฌ์„ฑํ’ˆ: Extraction buffer 50/25 mL (4์ข…), DNase 0.25 mL, Protease Inhibitor 1.25 mL

 

๋™๋ฌผ์„ธํฌ ๋‹จ๋ฐฑ์งˆ ์ถ”์ถœ Kit (ํ•ต, ๋ฏธํ† ์ฝ˜๋“œ๋ฆฌ์•„, ์„ธํฌ์งˆ ๋ถ„ํš์šฉ)
WSE-7422 EzSubcell Fraction

๋™๋ฌผ์„ธํฌ์™€ ํ˜ผํ•ฉํ•˜๋Š” ๊ฒƒ๋งŒ์œผ๋กœ ํ•ต, ๋ฏธํ† ์ฝ˜๋“œ๋ฆฌ์•„, ์„ธํฌ์งˆ๋กœ ๋ถ„ํšํ•  ์ˆ˜ ์žˆ๋Š” Kit์ž…๋‹ˆ๋‹ค. ์ถ”์ถœํ•œ ๋‹จ๋ฐฑ์งˆ์€ ์œ„์™€ ๋™์ผํ•˜๊ฒŒ ๋‹ค์–‘ํ•œ ์šฉ๋„๋กœ ์‚ฌ์šฉ์ด ๊ฐ€๋Šฅํ•ฉ๋‹ˆ๋‹ค.
ํ‚คํŠธ ๊ตฌ์„ฑํ’ˆ: Fraction buffer 50 mL (2์ข…), RIPA Lysis buffer 20 mL, Detergent mix 1 mL, Protease Inhibitor 0.7 mL

 

Phosphate buffer
WSE-7430 EzPBS

์„ธํฌ ์ค€๋น„์‹œ์— ์‚ฌ์šฉํ•˜๋Š” ์ผ๋ฐ˜์ ์ธ PBS Buffer์˜ 10๋ฐฐ ๋†์ถ•์šฉ์•ก์ž…๋‹ˆ๋‹ค.