This is a cached page for the URL (http://www.biotech.unl.edu/proteomics/protocol.html). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Protocols --Proteomics
Proteomics Core Facility

General Info

Service

Service Charges

Protocol

Research

Facility Advisory Group

Links



Protocol for Manual Peptide Synthesis

I. Resin Swelling And Coupling Of Activated Amino Acid Esters.
  1. Always wear gloves and observe good lab practices.
  2. Place a frit in a 3 ml syringe and push down with a pasteur pipette. make sure the frit is firmly in place.
  3. Weigh out 10 to 25 milligrams of solid support and pour into syringe. the amount depends on the level of substitution.
  4. Place the syringe firmly on the vacuum manifold.
  5. Turn the stopcock shut
  6. Using the dmf wash bottle, squirt approximately 0.5 ml on top of the support. release flow and allow to drain.
  7. Close flow control knob and add another aliquot of dmf to completely cover the support. let it stand for 10 minutes.
  8. Open flow and let dmf drain. close flow control knob.
  9. Add 0.25 ml of piperidine/dmf to support. allow to drain. close flow. add a further 0.25 ml of piperidine/dmf. let it stand for 5 minutes. drain.
  10. Wash resin with 5 x 1 ml of dmf with the flow open. shut flow valve.
  11. Dissolve the first amino acid-opfp ester in 0.25 ml of hobt/dmf solution by vortexing. (1g hobt/20 ml dmf)
  12. Add dissolved amino acid ester to resin, and let stand for 45 to 60 minutes with occasional mixing. drain.
  13. Wash resin with 5 x 1 ml of dmf with the flow valve open. shut flow valve.
  14. Start with step 9. after all the amino acids are coupled, remove the terminal fmoc-group with piperidine/dmf. wash the resin with dmf. finally wash the resin with 5 x 1 ml of dcm with the valve open. remove the syringe and dry overnight over p205. you are now ready to deblock and cleave the peptide.
II. Deblocking and cleaving peptide from solid support. All reactions are to be performed in a chemical hood
  1. Transfer the dried resin to a 1.5 ml microfuge tube, keep the syringe with the frit. take care to label each tube, so you know which peptide you are collecting.
  2. Add approximately 0.5 ml of deblocking solution to the syringe with a pasteur pipette. Make certain that the resin is completely submerged by the deblocking solution. (This solution is very caustic and toxic and will be prepared by Dr. Sarath.). Cap the tube and wrap the closure with a strip of parafilm and allow to deblock for 3 to 4 hours.
  3. After deblocking is complete filter the solution into 15 ml polypropylene tubes containing 10 ml of cold t-butylmethyl ether using the syringe + frit. Wash the resin with two 0.5 ml aliquots of neat tfa (caustic and toxic) and collect in the ether.
  4. Close the 15 ml pp tubes containing precipitated peptide (white flocculent material) with a single layer of parafilm and place in the -20c freezer for 30 min. Remove the tubes from the freezer, remove the parafilm cover and place the tubes in a clinical centrifuge in the hood. Centrifuge at top speed for 10 minutes. pour the solution into the waste bottle (labelled and in the hood). Resuspend the precipitate in 1-2 ml of 10 % acetic acid by vortexing with the cap on. Place tubes in the -20c freezer. Someone from the pcf will help you hard-freeze the material and show you the operation of the lyophilizer.
  5. Some peptides will not precipitate in ether.In these instances, collect the deblocked and cleaved material into 3 ml pp tubes, and remove the liquid on a centrifugal-evaporator (4-6 hrs). Resuspend the resinous material in 1 ml of 20 % acetic acid and place in the -20c freezer prior to lyophilization. Someone from the PCF will help you with the concentrator as well as the lyophilizer.
  6. Relyophilize the crude material after solubilization in 10 to 20 % acetic acid. Remove insolubles by centrifugation. Transfer the soluble fraction into a new tube, hard-freeze and lyophilize.This is the peptide fraction. Analyze by HPLC and or mass spectrometry.