• Created by Unknown User (lylaatta@mit.edu), last modified by Unknown User (teague@mit.edu) on Sep 24, 2014 14:35

Derived from the Abcam website: http://www.abcam.com/amyloid-beta-peptide-1-42-human-ab120301.html  This protocol is for 100 µg polypeptide.

  1. In a fume hood, resuspend 100 µg polypeptide in 100 µL neat HFIP (1,1,1,3,3,3-hexafluoro-2-propanol).
  2. Incubate at room temperature for 1 hour, vortexing briefly every 10 minutes at a moderate speed.
  3. Sonicate in a sonicating water bath on high for 10 minutes.
  4. In a fume hood, place the HFIP/peptide solution under a gentle stream of dry nitrogen gas.  Continue until the HFIP evaporates.
  5. Resuspend in 83 µl neat DMSO.  Incubate at room temperature for 12 minutes.
  6. Aliquot 10 µl volumes into individual 700 µl microcentrifuge tubes.  Store at -80°C.

Oligomerization

  1. Retrieve a single 10 µl aliquot from the -80.  Resuspend in 90 µl PBS for 100 µl total solution at a final concentration of 25 µM. 
  2. Incubate 2 hours at room temperature to allow for peptide aggregation.  
  3. If necessary, remove the insoluble portion by centrifuging at 16,000 xg for 15 minutes.
  4. The solution should be stable at 4° for at least a week.

    NOTE: The non-biotinylated ABetan (white tubes, labelled A{Beta}) is in 20 µl aliquots; resuspend in 180 µl PBS for the same working concentration. 

As per the LilrB2 paper , fig 1H, LilrB2/Beta amyloid binding gets close to saturation at about 500 nM.  This is 10 µl of 25 µM working stock diluted into 500 µl of media (in one well of a 24-well plate.)

 

 

Reconstituting Peptide from Package:

  1. Add 1% NH4OH directly to powder. Add 70-80 uL NH4OH per 1 mg of the peptide. 
  2. Dilute this solution in 1xPBS to your working concentration (should be less than 1mg/mL).
  3. Gently vortex to mix. 

Oligomerizing (LilrB2 Paper)

  1. Sonicate for 30 seconds (
  2. Dilute in 1X PBS to a concentration of 100 uM
  3. Incubate at 22C for 16 hours 
  4. Incubate at 4C for 24 hours
  5. Centrifuge at 16000 x g for 15 minutes 
  6. Collect the supernatant as oligomerized 
Oligomerizing (Other protocols)
  1. HFIP Treatment - to ensure monomerization (Abcam protocol)
    http://www.abcam.com/amyloid-beta-peptide-1-42-human-ab120301.html
    1. Dissolve peptide in 100% HFIP. Final concentration = 1mg/mL.
    2. Incubate at room temperature for 1 hour with occasional vortexing.
    3. Sonicate for 10 minutes in a water bath sonicator (optional according to some protocols).
    4. Aliquot into microcentrifuge tubes.
    5. Dry HFIP-peptide solution under a gentle stream of N2 gas (some protocols leave it to dry in a fume hood - no N2). 
    6. Store at -80C. Do not freeze thaw.
  2. Dissolve an aliquot in 100% DMSO to 5mM.
  3. Add to ice-cold F12 medium (DMEM?) to a concentration of 100 uM.
  4. Incubate at 4C for 24 hours. 
  5. Centrifuge at 14000 x g for 10 minutes. Supernatant is a mixture of monomers and oligomers. 

This protocol (steps 2-5) is primarily from a paper cited by several other papers, including (indirectly) the LilrB2 paper:

http://onlinelibrary.wiley.com/enhanced/doi/10.1046/j.1471-4159.2001.00592.x/

(HFIP monomerizes http://www.anaspec.com/products/product.asp?id=48257)


Verifying Identity of Supernatant: 

Western blot 

How much beta amyloid per aliquot?

100 µl aliquot size * 25 µmol/L beta amyloid concentration * (L/10^6 µl) * (1000 nmol / µmol) = 2.5 nmol / aliquot

 

How many µg is that?

2.5 nmol * (4853.9 g/mol) * (mol/10^9 nmol) * (10^6 µg/g) = 12.1 µg in 10 µL DMSO.

So, 100 µg gets us 8 aliquots of 12.1 ug in 80 µl DMSO.

 

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