Can anyone help me with construction of plasmid PFO/D4-GFP described by Rudolf Martin in IOVS 2014?
The authors describe the PFO/D4-GFP construct yet indicate GST during purification of this recombinant protein. They never described where they put GST- before PFO/D4 or after GFP? Overall, the description of the construct is very vague with a lot of details omitting. Any help on preparation of this construct will be highly appreciated.
This construct will be very useful for detection of both un-esterified as well as esterified cholesterol and will be best alternative to Filipin staining which is very hard to work with (auto-bleach very very quickly specially during images acquisition.
thank you
Aicha
Hi Aicha-
People can guess how it might have been constructed, but the only people that really know are the authors of the paper. Unfortunately they were not funded by the NIH and I didn't see anything in the journal policies explicitly requiring them to provide the plasmid to anyone who asks. However, it is generally continued good practice/scientific ethics to AT LEAST accurately describe the construct and, ideally, also provide a small amount of the plasmid. Try writing the corresponding author a brief, polite email complimenting their paper and asking for more details and/or a sample of their plasmid (you could offer to pay for shipping).
This paper has information on how people cloned and expressed the fragment, labelled with an Alexa fluor, and then used it in fixed cells (in case that would be useful):
Lipids and Lipoproteins: Metabolism,
doi: 10.1074/jbc.M800440200 originally published online April 29, 2008
Hi Amanda and thank you for taking a time to share your thoughts. We tried to contacted the authors, unfortunately they stopped answering our multiple questions or should I say clarifications?. This is unfortunate because what is the meaning of our publications and findings if no body can replicate them and benefit from them??? We could go on and on about scientific ethics, but I have fate that the authors might, one day, decide to share with others as we are not the only lab that acquired information about this construct.
aicha
Alas! Here are some things to try:
1. Just make a fresh start and make sure your questions are entirely clear.
Perhaps even try a phone call? Also it never hurts to be diplomatic - might as well heap on praise about how wonderful and great their research is. You could also offer a collaboration to entice them.
2. Have your PI or department head send the email instead of you with clear indication of how important they are.
3. If no response, have the important person include strong language about ethics and/or contacting the journal where it was published.
4. Contact the editors of the journal to see if this matter concerns them and/or if they would help encourage the authors.
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