Paul.xu at 17:53, 12 September 2019

2019-09-12T17:53:33Z

New page

2014/3/

[[File:2115 1315 1592 1996.jpg|RTENOTITLE]]

{| width="200" border="1" cellspacing="1" cellpadding="1"
|-
| lane 
| colspan="5" | 1 
| colspan="2" | 2 
| 3 
| 4 
| colspan="2" | 5 
| 
| colspan="3" | 6 
| 7 
| 8 
|-
| clone ID
| colspan="11" | 2115, MW is 52.8 kDa
pT7A3-iFLP

| PageRuler Plus Prestained protein ladder 
| colspan="2" | pL6H-LambdaExo 
| colspan="2" | pT7A3-Ecoli-Topo_I 
| pT7A3-MthRnl  
|-
| host 
| colspan="11" | Nico21DE3plysN 
| 
| colspan="5" | Nico21DE3plysN 
|-
| induction 
| colspan="11" | 0.1 mM IPTG 25 oC o/n 
| 
| colspan="5" | 0.2 mM IPTG 25 oC o/n 
|-
| purification 
| colspan="11" | Ni beads 
| 
| colspan="5" | Ni beads 
|-
| lysis buffer 
| colspan="3" | buffer A ---- 99oC 10min 
| colspan="2" | buffer A 
| colspan="2" | buffer A (containing 1M Nacl) 
| colspan="2" | purification lysis buffer(containing NP40+1M Nacl) 
| colspan="2" | expression screening lysis buffer(containing 4M Urea+1% SDS) 
| 
| colspan="5" | 
|-
| MW 
| colspan="11" | pI/Mw: 9.76 / 52.8 
| 
| colspan="3" | 6.06 / 27.1 
| 8.68/97.3 
| 5.81 / 45.3 
|}

2115 pT7A3-iFLP----8L, Nico(DE3)plysN, 0.1mM IPTG RT induce overnight, 34.1g use 5ml induced culture to chose the best lysis buffer 5. expression screening lysis buffer(containing 4M Urea+1% SDS) -------clear----have expression 4. purification lysis buffer(containing NP40+1M Nacl)-------------white pellet-------no expression 3. buffer A (containing 1M Nacl)-------------white pellet-------no expression 2. buffer A -------------white pellet-------no expression 1. buffer A ---- 99oC 10min---------white pellet-------no expression

then add buffer A containing 1M Nacl to pellets 1.add Urea to final concentration of 4M------no change 2.add SDS to final concentration of 1 % -------half of the pellet dissolved 3.add Urea to final concentration of 4M and add SDS to final concentration of 1 % ------ all of the pellet dissolved 4.add Arginine to final concentration of 0.1M, add glycerol to to final concentration of 10 %, add triton-x100 to final concentration of 0.5%-----no change add lysis buffer (containing DnaseI+ lysozyme) to pellet-----37oC 2hours----no change

[[Category:Protein Purification]]

2115 1996 1592 1315 - Revision history

Paul.xu at 17:53, 12 September 2019