Editing 2115 1996 1592 1315

<br/>2014/3/

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



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

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



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

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




[[Category:Protein Purification]]