Fluorescent Recovery After Photobleaching (FRAP) is an optical technique that allows you to identify the optimal conditions for LCP crystal growth without having to wait on the crystals actually growing. The automated process in this imager can analyze a 96-well plate in less than 45 minutes.
Find the optimal conditions for LCP crystal growth without waiting.
FRAP (Fluorescence Recovery After Photobleaching) imaging was developed at Scripps Institute to advance Lipidic Cubic Phase (LCP) condition screening, often a time-consuming process that involves waiting for days or weeks to see if experimental conditions support crystal growth. The commercial FRAP imager is produced exclusively by Formulatrix. FRAP is most useful for studying G protein coupled receptor (GPCR) proteins.
What is FRAP?
FRAP is the process of tagging a protein with a fluorescent marker, using a laser to bleach a small part of the LCP drop, and repeatedly photographing the drop to monitor how long it takes for uniform fluorescence to recover.
High Throughput FRAP
Given the time required for the protein to recover from bleaching, FRAP can be a time-consuming process. To expedite the FRAP process, the FRAP imager can be used to bleach and photograph a 96-well LCP plate in under 50 minutes using high throughput settings.
Using this method, the imager executes a drop location and auto focus algorithm on each drop and takes beginning state images, then bleaches each of the 96 LCP drops sequentially and takes “time zero” images, and finally returns to the first drop and photographs the bleached drop´s activity at a pre-configured end-time.
The resulting FRAP mobile-fraction score, viewable in the Rock Maker software, is enough to determine whether well conditions are amenable to crystallization.
Full Recovery FRAP
Drops imaged using the full recovery setting will be photographed many more times following the bleach time (time zero) in order to more closely capture molecular diffusion and generate a single- or double-component Bessel function curve. Single-component recovery curves are enough for most experiments to analyze a protein´s diffusion rate and recovery time. However, if the experiment has two different types of molecules diffusing, such as labelled lipids, then a double-component Bessel function curve is more appropriate.
One of the main factors for successful LCP crystallization is the ability of the protein to diffuse within the lipid bilayer. The diffusion rate of the protein is influenced by protein aggregation, structural properties of the LCP, and the chemical environment. The diffusion rate can be determined by FRAP, which measures the amount of time required for the fluorescence intensity of a tagged protein to reestablish itself within a small area in the LCP drop that has been subject to optical bleaching. Using FRAP you can screen your crystallization experiment in under 50 minutes to determine the mobile fraction of your protein and whether or not that condition is conducive to forming protein crystals.
FRAP automatically calculates the percent mobile fraction for each drop. The mobile fraction score can then be related to the ability of the protein in that condition to form protein crystals. The higher the mobility, the greater the chance for crystallization. Scores are displayed in a color coded over-view allowing users to identify positive wells at a glance.
Users can mark positive wells in the software for future reference. In the example to the left, well H8 has the highest mobile fraction rate and is most likely to grow crystals. Well D12 has no mobile fraction and is the least likely to grow crystals.
Rock Imager 1000 can be expanded to include FRAP in the base of its chassis. The dual imager setup has the complete functionality of a FRAP benchtop imager and also includes an automated visible imager for routine imaging of crystallization plates. Rock Imager can store and incubate up to 1,000 plates and can image plates in the visible imager while FRAP experiments are performed in the FRAP cabinet. UV imaging can also be included in the setup.
Image quality is retained even with fans circulating, motors running and air flowing. Without our dedicated design towards minimizing vibration, image quality would be severely affected resulting in poor image quality despite good optics. Innovative mechanical design, advanced vibration damping materials, and special motor tuning ensure extremely low vibration to conserve image quality and minimize disturbance to the protein drops.
Recent advances in automation technology for drop setting, condition screening and imaging make LCP crystallization considerably more accessible. This application note discusses the three major steps of LCP crystallization, including FRAP, and how automation technology accelerates LCP crystallography.
A high protein mobile fraction and a fast diffusion rate correlate very well with known crystallization conditions. The FRAP imager bleaches the dye-labeled protein in LCP with a laser pulse. Then a sequence of post-bleach images are recorded. The image processing function in the imager software measures the pixel intensity inside the bleached spot. Protein mobility parameters are extracted from fitting the pixel intensity recovery curve with either a single or double component 2D diffusion equation. The mobile fraction can then be used to determine if the crystallization condition ia conductive to forming protein crystals
Compatible Plate Types:
|Zoom||N.A.||Depth of Field||Field of view||Pixel size|
|1.1x||0.06||294 μm||8.1mm x 6.1mm||5.8 μm|
|5.5x||0.25||17 μm||1.6mm x 1.2mm||1.2 μm|
Gustavo Fenalti, Enrique E. Abola, Chong Wang, Beili Wu, Vadim Cherezov (2015). Chapter Twenty – Fluorescence Recovery After Photobleaching in Lipidic Cubic Phase (LCP-FRAP): A Precrystallization Assay for Membrane Proteins Methods in Enzymology 557: 417–437
V, Liu J, Hanson MA, Griffith MT and Stevens RC (2008) LCP-FRAP assay for pre-screening membrane proteins for in meso crystallization. Cryst Growth Des 8: 4307-4315
Xu F., Liu W, Hanson MA, Stevens RC and Cherezov V (2011) Development of an automated high throughput LCP-FRAP assay to guide membrane protein crystallization in lipid mesophases. Cryst Growth Des 11: 1193-1201
Average User Satisfaction Rate
|A leading Chinese academic lab - Wenru Zhang||8/11/2014 11:40 PM|
FRAP (Fluorescence recovery after photobleaching) measures membrane protein mobility in lipids in a high throughput fashion. The system utilizes an automated high throughput LCP-FRAP assay to prescreen membrane protein crystallization conditions.
|Shanghai Institute of Materia Medica - Qiang Zhao||7/24/2014 4:54 PM|
The RI1000 and FRAP are almost perfect. Very stable, very productive. Software is easy to follow, and the maintain service is good and effective. Prices are a little higher than expected, but I have to say it worth it.
|Merck - Sangita Patel||10/20/2014 11:40 PM|
Getting it soon, can't wait to put my hands on it.