Paul.xu at 23:20, 30 January 2014

2014-01-30T23:20:01Z

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== Expression of yFlp at 18 oC degree ==

Transform plasmid with BL21-AI/pRare2LysS. Induce with 0.1% arabinose and 0.15 mM IPTG, 18 oC ~18 hours. 

Spin down 5 ml, lyse, pulldown using Ni beads. 

{| width="301" cellspacing="1" cellpadding="1" border="1"
|-
| 
| Clone ID 
| Plasmid Name 
|-
| 1 
| 1870 
| pET28b-HTNFlp-ecoli 4oc 
|-
| 2 
| 1870 
| pET28b-HTNFlp-ecoli 18oc 
|-
| 3 
| 1853 
| pET28b-yFLP 4oc 
|-
| 4 
| 1853 
| pET28b-yFLP 18oc 
|-
| 5 
| colspan="2" | Fementas PageRuler Plus
|-
| 6 &7 
| 
| 
|}

 

[[Image:Flp expression at 18 degree 1 2014.jpg]] 

 

== Use chaperones to improve Flp expression ==

1/25/2014 

Test chaperone on Tat-yFLP expression 

[[Image:Chaperone on FLP expression 1 25 14.jpg]]

 

{| width="832" cellspacing="1" cellpadding="1" border="1"
|-
| Lane 
| Name 
| Protein Name 
| Clone ID 
| Host 
| Induction 
| Purification 
| pI/Mw 
|-
| 1 
| pET28b-yFLP 
| Tat-yFLP 
| 1853 
|
5 ml, 

Tuner(DE3)-pG-KJE8

| 0.5 mM IPTG  RT o/n 
|
Ni beads

|
9.76 / 52.8

|-
| 2 
| pET28b-yFLP 
| Tat-yFLP 
| 1853 
|
 5 ml,

Tuner(DE3)-pG-KJE7 

| 0.5 mM IPTG RT o/n 
|
Ni beads

|
9.76 / 52.8 

|-
| 3 
| pET28b-yFLP 
| Tat-yFLP 
| 1853 
|
5 ml,

Tuner(DE3)-pTf16 

| 0.5 mM IPTG RT o/n 
|
Ni beads

|
9.76 / 52.8

|-
| 4 
| pCold-Tat-yFLP 
| Tat-yFLP 
| 2031 
|
5 ml,

Tuner(DE3) 

| 0.5 mM IPTG RT o/n 
|
Ni  beads

|
9.76 / 52.8

|-
| 5 
| Fermentas PageRuler Plus 
| 
| 
| 
| 
| 
| 
|-
| 6 
| pCold-Tat-yFLP 
| Tat-yFLP 
| 2031 
| 5 ml,
Tuner(DE3)-pG-KJE8 

| 0.5 mM IPTG RT o/n 
|
 

Ni beads

|
9.76 / 52.8 

|-
| 7 
| pCold-Tat-yFLP 
| Tat-yFLP 
| 2031 
|   5 ml,
Tuner(DE3)-pG-KJE7

 

| 0.5 mM IPTG RT o/n 
|
Ni beads

|
9.76 / 52.8

|-
| 8 
| pCold-Tat-yFLP 
| Tat-yFLP 
| 2031 
|  5 ml,
Tuner(DE3)-pTf2

 

| 0.5 mM IPTG RT o/n 
| Ni beads 
|
9.76 / 52.8 

|-
| 9 
| pCold-Tat-yFLP 
| Tat-yFLP 
| 2031 
|   5 ml,
Tuner(DE3)-pTf16

 

|  0.5 mM IPTG RT o/n 
|
Ni beads

|
9.76 / 52.8

 

|-
| 10 
| pET28b-yFLP 
| Tat-yFlp
| 1853 
| 5 ml,
Tuner(DE3)-pTf2 

|   0.5mM IPTG RT o/n 
| Ni beads 
| 9.76 / 52.8 
|-
| 11 
| pET28b-yFLP 
| Tat-yFlp 
| 1853 
| BL21-AI 
| 0.5mM IPTG + 0.1%arabinose RT o/n 
| Ni beads 
| 9.76 / 52.8 
|}

 

Conclusion: Chaperones do not enhance protein expression. Protein is expressed as truncated. Less than 53 kDa 

== Expression in pT7A3 and pCold ==

[[Image:FLP_exp_BL21_strains_1_2014.jpg]]

== Other Protein Expression Screening ==

[[Image:1 24 14 protein exp screening 1.jpg]]

[[Image:1 24 14 protein exp screening 2.jpg]]

[[Category:Protein_Purification]]

1853 2031 - Revision history

Paul.xu at 23:20, 30 January 2014