Soluble all ligaments of amyloid genic proteins through their cytotoxic properties are believed to underlie the pathophysiology of neurodegenerative disease. These all ligaments form and dissociate continuously when in solution. A fact that complicates the structural studies that are critical for developing inhibitory molecules to circumvent this problem.
Photo induced cross-linking of unmodified proteins or pickup is used. This method involves the exposure of disaggregated and solubilized, amyloid genic peptides to cross-linking reagents and a one second pulse of intense light to covalently link dimers and allers. The allers size distribution can then be analyzed via standard biochemical techniques.
Hi, I am far Rahimi from the Batan Laboratory in the Department of Neurology at the University of California in Los Angeles. I am Al Mai from the Bean Laboratory. Today we'll show you a procedure for photo induced crosslinking of unmodified proteins.
We use this procedure in our laboratory to generate stabilized amyloid beta protein oligomers and study the I picked up structural modifications on the oligo high distribution. This procedure was initially published by Fancy and Coate to study stable protein complexes. The method was applied later by Battan and others to quantitative study of meta stable amyloid protein assemblies, including a beta pram and alpha sycle.
So let's get started. To begin the protocol HEXA fluoro ISOPROPANOL or HFIP, which has been pre chilled on ice, is added to a silicon coated low absorbent tube containing one to 200 micrograms of lyophilized peptide. So that the final concentration is about 0.5.
Millimolar HFIP prevents amyloid genic peptides from aggregating and will improve the consistency of the results. Now sonicate the peptide tube for five minutes. At room temperature.
When sonication is complete, gently vortex the tubes and allow them to incubate for 30 minutes at room temperature. After 30 minutes, chill the peptides on ice for one minute. The chilled peptides can now be divided into 10 to 50 microliter aliquots and 0.6 mil low absorbent tubes.
Next place the tubes in the hood with the caps off. To allow the HFIP to evaporate overnight, wrap the rack of tubes and Kim wipes to prevent dust from settling in the tubes. Once most of the HFIP has evaporated any trace HFIP must be removed by placing the tubes in an ex under vacuum for two hours.
The final product is a thin layer of peptide film within the tubes. These tubes can be stored for months at minus 20 or minus 80 degrees Celsius. To begin the solubilizing and photo cross linking steps, the peptide film is first dissolved in a solution of dilute sodium hydroxide.
After adding the sodium hydroxide, deionized water and then phosphate buffer is added, the final solution should be 45%phosphate buffer, 45%water, and 10%sodium hydroxide solution. If residual protein film is left on the side of the tube, scrape it into the solution. Using a pipette tip and pipette up and down the suspended peptides should now be sonicated in a water bath Sonica sonicate for one to two minutes.
After sonication, add the phosphate solution and mix by pipetting up and down. Then centrifuge the tube at 16, 000 G for 10 minutes. While the centrifuge is spinning, prepare your camera for the photo cross linking steps.
The camera shutter delay should be set to one second and the shutter should be loaded. Once the tubes have finished centrifuging make 18 microliter aliquots of the peptide solution in clear 0.2 mil PCR tubes. These aliquots will be used for the photo crosslinking.
Be sure to set aside some peptide solution to be used as a negative control just before crosslinking. Add one microliter of ammonium per sulfate and one microliter of ruthenium. Now quickly place the tube inside a 1.8 M glass vial and place the vial inside the bellows attached to the front of the camera body.
Close the camera with the lens protector and press the shutter. The sample is now irradiated for one second. At higher irradiation times extensive radical reactions cause protein degradation.
Once irradiated, quickly recover the 0.2 mil PCR tube and add 10 microliters of pre-prepared page sample buffer containing Beta mer capto ethanol. The cross-linked peptides can be stored for up to seven days at minus 20 degrees Celsius. Prior to SDS page, fractionation and silver staining.
To examine the cross-linked peptides routine SDS page and silver staining may be used in estimating the volumes you should load five microliters of the reaction mixture should yield roughly 130 picomoles of peptide. It is recommended to use the Xcel Sherlock mini cell system from in Vitrogen for gel electrophoresis and to perform silver staining. According to in Vitrogen publication, IM 1 0 0 2, no NOx precast gel electrophoresis guide analyze your SDS page gel after silver staining, the uncross linked control polypeptide, GRF runs mainly as a monomer.
Cross-linked GRF produces continuous all liga distribution ranging from dimer through hexamer such that the apparent relative band intensity of oligomers decreases exponentially with increasing molecular mass. Un cross-linked calcitonin mainly runs as a monomer and some as a dimer. Cross-linked calcitonin yields an a liga size distribution that strongly diverges from a monotonous exponential decrease in the region.
Monomer through tetramer suggesting pre-existence of dimers trimmers and tetramers un cross-linked. A beta 40 runs only as a monomer pickup generated a Beta 40. All ligaments show approximately similar band intensities of monomer through tetramer, followed by a decrease in the abundance of higher all ligaments.
Un cross-linked. A beta 42 produces predominantly two bands, a monomer band and a trier temer band. The Trier Tetramer Band is an artifact produced by SDS Cross-Linked.
A Beta 42 shows a distinct all lir size distribution comprising two groups. The first group monomer through trier displays decreasing intensity with increasing allmer order. In the second group, a gian like distribution is observed between Teer and Hep Tor with a maximum at Pentamer and Hexamer.
We have just so you how to cross-link the protein using pika Using this procedure. It's important to remember that when we are using amyloid genic protein, we have to make sure that the preparations are aggregate free to obtain reproducible results, the conditions shown here, and these experiments can be modified experimentally according to the lytes of interest. So that's it.
Thanks for watching and good luck using pickup experiment.
