Difference between revisions of "Part:BBa K243032"
(→Detection) |
(→Detection) |
||
| Line 11: | Line 11: | ||
[[Image:Freiburg RV308 mit fluo Oligos3 (c1+c2).JPG|350x250px]]<br> | [[Image:Freiburg RV308 mit fluo Oligos3 (c1+c2).JPG|350x250px]]<br> | ||
| − | The cell extract were check by SDS-page to get information about the expression of proteins.<br> | + | The cell extract were check by SDS-page to get information about the expression of proteins.<br><br> |
[[Image:SDSfokvenus.jpg]]<br> | [[Image:SDSfokvenus.jpg]]<br> | ||
---- | ---- | ||
SDS gel; pEx Venus-Fok_a in BL21de3; lanes: Marker (ColorPlus Prestained Protein Marker, Broad Range), clone 1 uninduced, clone 1 induced, clone 1 induced, clone 2 uninduced, clone 2 induced, control (His-FluA-SplitLi-Fok_i + His-Dig-SplitLi-Fok_a) uninduced, control induced) | SDS gel; pEx Venus-Fok_a in BL21de3; lanes: Marker (ColorPlus Prestained Protein Marker, Broad Range), clone 1 uninduced, clone 1 induced, clone 1 induced, clone 2 uninduced, clone 2 induced, control (His-FluA-SplitLi-Fok_i + His-Dig-SplitLi-Fok_a) uninduced, control induced) | ||
---- | ---- | ||
| − | In the lanes of the induced clones ( lanes 3,4) appears a band at about 50 kDA which could be the Venus-Fok_a construct.<br>> | + | In the lanes of the induced clones ( lanes 3,4) appears a band at about 50 kDA which could be the Venus-Fok_a construct.<br><br> |
| − | A western blot with specific anti-GFP antibodies (first antibody: Santa Cruz Biotechnology sc-9996 mouse αGFP; second antibody: Santa Cruz Biotechnology sc-2005 goat αmouse IgG-HRP)was conducted to proof the complete expression of the protein+YFP construct and to excluded a degradation of | + | A western blot with specific anti-GFP antibodies (first antibody: Santa Cruz Biotechnology sc-9996 mouse αGFP; second antibody: Santa Cruz Biotechnology sc-2005 goat αmouse IgG-HRP)was conducted to proof the complete expression of the protein+YFP construct and to excluded a degradation of the construct.<br><br> |
| − | [[Image:Freiburg09 WBfokvenus.jpg]] | + | [[Image:Freiburg09 WBfokvenus.jpg]]<br> |
===Purification=== | ===Purification=== | ||
Revision as of 19:26, 21 October 2009
Fok_a-Venus
This part is like part the protein domain Fok_a from our universal endonuclease, but it has an additional linked YFP-marker.
Usage and Biology
The addition of a fluorescent protein(Venus) to the protein construct, enables the detection by fluorescence microscope and new way of purification by GFP-trap column.The Venus protein was fusioned C'terminal to the protein domain, so when the protein is expressed the fluorescence signal of the Venus protein can be seen.
Detection
There was a strong fluorescence signal under the fluorescence microscope after the induction of the cells with IPTG.The E.colis(RV308) were excited with the wavelength 505nm and then some pictures of the fluorescencewere made.
.JPG)
The cell extract were check by SDS-page to get information about the expression of proteins.
SDS gel; pEx Venus-Fok_a in BL21de3; lanes: Marker (ColorPlus Prestained Protein Marker, Broad Range), clone 1 uninduced, clone 1 induced, clone 1 induced, clone 2 uninduced, clone 2 induced, control (His-FluA-SplitLi-Fok_i + His-Dig-SplitLi-Fok_a) uninduced, control induced)
In the lanes of the induced clones ( lanes 3,4) appears a band at about 50 kDA which could be the Venus-Fok_a construct.
A western blot with specific anti-GFP antibodies (first antibody: Santa Cruz Biotechnology sc-9996 mouse αGFP; second antibody: Santa Cruz Biotechnology sc-2005 goat αmouse IgG-HRP)was conducted to proof the complete expression of the protein+YFP construct and to excluded a degradation of the construct.
Purification
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 487