Purif.

lacR pe HisTrap HP, 5ml (21-07-15):

Expresie: 4.5L in BL21(DE3)RIL
Centrif cultura in falcoane 50 ml 4h (este defecta centrifuga Beckman pt. volume mari)
Stocare pellet la -80C.
Resuspendare pellet in lysis buffer: 40 ml lysis b./ 1L cultura (180 ml/4.5L cultura)
Trecere prin potter 4 ori.
Tratare cu lizozim 0.5 mg/ml (Fluka) 1h la 4C.
Liza: French Press de 2 ori. Am oprit 20 microl. lizat total (LT) pentru SDS-PAGE.
Tratare cu DNase (Sigma) (de la Catalina) 0.5 mg/ml in prezenta de 0.5 mM MgCl2,
incubare 30 min la RT (25C).
Centrif: 50 min, 19000xg, 4C. SN l-am filtrat prin filtru 0.45 m. Oprit: 20 microl
solubil (S) si pellet (fractia insolubila).
Proba filtrata incarcata pe Superloop (160 ml).
Incarcare proba pe coloana: flow: 1ml/min. Oprit flow through (FT).
Spalare: 1.5 ml/min pana la A280m= 44 mAU
Elutie: 50%B in 40 min
Flow: 0.7 ml/min
Fractii: 0.5 ml/ fractie
Lysis buffer:
20 mM Tris-HCl, 500 mM NaCl, 0.5 mM DTT, 10 % glicerol , pH = 8.0 cu 1 mM PMSF,
inhibitori proteaza (EDTA-free)
Buffer A (Washing buffer): 20 mM Tris-HCl, 500 mM NaCl, 0.5 mM DTT, 10 % glicerol
, pH = 8.0 cu 10 mM imidazol
Buffer B (Elution buffer): buffer A cu 500 mM imidazol

Proba dupa expresie (pellet 1ml cultura): pellet + 50 l 2XLB

Solubil: 20 l + 20 l 2XLB
Pellet: reluat in 300 l 2XLB
FT: 25 l + 25 l 2XLB
Elutii: 25 l + 25 l 2XLB
Fractii colectate: Incarcat 15 l/gel

Gel 1 10%SDS-PAGE:
1. LMW
2. Proba dupa expr.
3. Pellet
4. Solubil
5. FT
6. fr. 10
7. 12
8. 14
9. 16
10. 18
11. 22
12. 26
13. 28
14. 30
15. 32
Gel 2 10% SDS-PAGE:
1. LMW
2. fr. 34
3. 36
4. 38
5. 40
6. 42
7. 44
8. 46
9. 50
10. 52
11. 56
12. 62
13. 70
14. 75
15. 80
Probele au stat peste noapte la 4C, fara dializa.
A doua zi, am reunit probele in trei pool-uri:
A. fr. 10 16 (3 ml)
B. fr. 17 31 (7.5ml)
C. fr. 32 83 (25 ml)
Proteina totala Bradford (Ana) (22.07.15):
A. fr. 10 16 (3 ml) conc. prot. 0.12 g/l (total: 0.365 mg/3ml)
B. fr. 17 31 (7ml) conc. prot. 1.698 g/l (total: 12.7 mg/7.5ml)
C. fr. 32 83 (25 ml) conc. prot. 0.43 g/l (total: 10.8 mg/25ml)