Biochemistry
A subscription to JoVE is required to view this content.
You will only be able to see the first 2 minutes.
The JoVE video player is compatible with HTML5 and Adobe Flash. Older browsers that do not support HTML5 and the H.264 video codec will still use a Flash-based video player. We recommend downloading the newest version of Flash here, but we support all versions 10 and above.
If that doesn't help, please let us know.
How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells
Chapters
Summary January 7th, 2019
Here, we present a protocol that involves genetically coupled spectrally distinct photoactivatable and fluorescent proteins. These fluorescent protein chimeras permit quantification of the PA-FP fraction that is photoactivated to be fluorescent, i.e., the photoactivation efficiency. The protocol reveals that different modes of photoactivation yield different photoactivation efficiencies.
Transcript
We present here a protocol that involves genetically coupled spectrally distinct photo-activatable and fluorescent proteins.These internal rulers permit quantification of the PAFP fraction that is photo activated to be fluorescent.Thus far, no method has been available to quantify in bulk in life cells how many of the PAFP expressed are photo activated to fluoresce.The presented quantification of photo-activation efficiency is exemplified for PA-GFP and PA Cherry in live cells but it is in principle broadly applicable and can be used for any photo-activatable fluorescent protein under any experimental condition.Whenever working with genetically encoded fluorescent proteins, the absolute amount of proteins expressed varies from cell to cell and it is unknown.To standardize the expression among cells, we introduce internal rulers of genetically coupled spectrally distinct fluorescent proteins.By coupling the genetic information of a photo-activatable florescent protein to a spectrally distinct always on fluorescent protein internal rulers are created that will still be expressed to an unknown total amount but in a fixed known relative amount of one to one.To begin construct the plasmids as described in the accompanying text protocol.Also, culture in the standard cell line in its perspective medium using the phenol red free version.When ready to begin the experiment, harvest the cells using trypsin without phenol red to reduce background florescence.After harvest, fill a 2 ml pipette with the cell-suspension from a confluent cell culture grown in a T 12.5 cell culture flask.Add one drop into each chamber of an eight chamber glass slide.Incubate the slide under standard cell culture conditions for 24 hours.Next transfect the cells using commercial reagents following the distributors protocol.Transfect the cells with the GFP Cherry, PA-GFP Cherry and GFPPA Cherry chimeras.Then return the cells to the incubator to allow for protein expression, folding, and maturation.After a total of 20 hours post transfection, transfer the cells to the stage of a confocal florescent microscope equipped with the humidified and heated environmental chamber pre-warmed to 37
Related Videos
You might already have access to this content!
Please enter your Institution or Company email below to check.
has access to
Please create a free JoVE account to get access
Login to access JoVE
Please login to your JoVE account to get access
We use/store this info to ensure you have proper access and that your account is secure. We may use this info to send you notifications about your account, your institutional access, and/or other related products. To learn more about our GDPR policies click here.
If you want more info regarding data storage, please contact gdpr@jove.com.
Please enter your email address so we may send you a link to reset your password.
We use/store this info to ensure you have proper access and that your account is secure. We may use this info to send you notifications about your account, your institutional access, and/or other related products. To learn more about our GDPR policies click here.
If you want more info regarding data storage, please contact gdpr@jove.com.
Your JoVE Unlimited Free Trial
Fill the form to request your free trial.
We use/store this info to ensure you have proper access and that your account is secure. We may use this info to send you notifications about your account, your institutional access, and/or other related products. To learn more about our GDPR policies click here.
If you want more info regarding data storage, please contact gdpr@jove.com.
Thank You!
A JoVE representative will be in touch with you shortly.
Thank You!
You have already requested a trial and a JoVE representative will be in touch with you shortly. If you need immediate assistance, please email us at subscriptions@jove.com.
Thank You!
Please enjoy a free 2-hour trial. In order to begin, please login.
Thank You!
You have unlocked a 2-hour free trial now. All JoVE videos and articles can be accessed for free.
To get started, a verification email has been sent to email@institution.com. Please follow the link in the email to activate your free trial account. If you do not see the message in your inbox, please check your "Spam" folder.
