I dissolved 0.1 mg of β-amyloid (1-42) in 0.1 ml of HFIP. After evaporation of HFIP by speedvac further it was dissolved in 1X PBS buffer (concentration= approximately 22 μM ). But when I was checking the Optical Density (Molar Extinction Coefficient = 1450 at 276 nm) to confirm the actual concentration, I found it 0.15897 which is corresponding to 109.63 μM of β-amyloid. I am also attaching the absorption spectrum of β-amyloid (1-42) which I was observed during my experiment. Please help me in this regard.
I don't know the exact amino acid sequence of the β-amyloid peptide, but the link I attached may help you to quantify the absorption spectrum.
You need to know the AA sequence. With Trp or Tyr inside, you'll have chance to calculate the right concentration by UV/VIS spectrometry (because these are the only AA which absorb specifically in UV and have a known extinction coefficient). You may need to add urea or guanidinium chloride to unfold the peptide (which I think, folds for sure, making UV/VIS without denauturation agents not very meaningful) - does the UV/VIS spectrum change in that case? You'll find the detailled description in the Sigma Aldrich link attached.
If you do not have any Trp or Tyr, this will be hard to determine. The spectrum looks like you would have some Trp or Tyr, but usually you have kind of peak in that case (I attached a spectrum to show you what I mean). You could change the concentration to improve the spectrum.
http://www.sigmaaldrich.com/life-science/custom-oligos/custom-peptides/learning-center/concentration-calculation.html
Since Abeta contains single Tyr a. a. at residue 10, our lab determine the concentration of Abeta by mea
The Theoretical Molar Extinction Coefficient that you are using is correct.
Your result is around half of the concentration you were expecting (~220uM). Is this right?
How certain are you of the 0.1 mg measurement? Because this is a rather small amount of protein and the error of its measurement can really make a huge difference. Given this small amount, for me it is not so strange that you came up with around half of what you were expecting. The procedure you are using to measure the protein is exactly the same I use and like that you can always control per each reconstitution what is the final working concentration for your experiments.
Best of luck!!
Hugo is right, you may start with a higher concentrated stock solution rather than weighting such small amounts of solid.
1 mg of commercially obtained amyloid peptide in a bottle can actually weight almost 2 mg, thus a 0.1 mg aliquot is 1/20th part of "1 mg of peptide" and your result is correct. Was the pathlenght 1 cm?
hi check wit Professor Kaj Blennow at Sahlgrenska University Hospital best Yvonne
You might want to check this publication: http://www.ncbi.nlm.nih.gov/pubmed/21565831
Besides HFIP, a DMSO treatment (combined with solvent removal, like desalting) might be required to entirely disrupt pre-existing aggregates. From the UV -signals in the 300nm-400nm range it seems you have some light scattering from the Abeta, which means it is not only monomer in your PBS solution. This will of course influence your concentration read-out. Please also beware that the formation of a dityrosine link might lead to a significantly increased epsylon280nm value for the dityrosine linked peptide (and an SDS-PAGE would certainly reveal the presence of such covalent dimers). Also try to workl as much on ice as possible with your freshly dissolved peptide sample, to avoid aggregation and reduce as much as possible the time between dissolving the peptide and measuring its concentration.
Good-luck!
Using the extinction coefficient to measure the concentration of the amyloid peptide is somewhat tricky. When the monomeric peptide aggregates into fibres the extinction increases because the fibers are scattering the light which mimics a higher absorption.
After starting the aggregation process you will lose the control over that peptide. The fibres may stick to surfaces and may be lost. Or the extinction shoes you crazy numbers.
With best regards,
Heinz
The problem is light scattering from large aggregates as Kris suggested. The absorbance spectrum from an aggregate free solution of Abeta should have a clear peak near 276 with an absorbance near zero in the wavelengths around it after the blank sample is subtracted.
To remove aggregates:
1. Run the Speedvac for at least a day to remove HFIP. Or better yet, lyophillize it for a day. Even small amounts of residual HFIP can cause significant can cause significant problems with reproducibility and alteration in mechanism.
2. After the film is dried, dissolve it in either DMSO or 1 mM NaOH (ph 10) at 10x the concentration. We prefer the NaOH method as DMSO can interfere with some biophysical methods, suppresses free radical generation, and can interfere with some cell viability methods. However, DMSO solutions are more stable.
Some other points:
1.Commercial peptides are usually sold by total weight, not peptide content. A 1 mg bottle is usually 0.4-0.8 mg peptide.This amount can vary substantially from manufacturer and from lot to lot.
2. Stock solutions should not be kept if possible. Split the sample first and store the dried films instead.
There is a running thread on these types of issues here
https://www.researchgate.net/post/Amyloid_protocols_project?_tpcectx=profile_questions
and
https://www.researchgate.net/project/Amyloid_open_protocols
Thank you to all Researcher for valuable suggestions I shall try as you advised
@Marcus Binner beta amyloid contains 1 tyr and 3 phe so molar extinction coefficient is very low
@Hugo Vicente Miranda I understand the problem (the amount of protein is very low) but i am expecting the concentration is 22uM not 220uM
Hi Madhusudan,
If you dissolve the Abeta peptide film after HFIP treatment in 1 mM NaOH and you dilute it out in a buffer with sufficient buffering capacity, you can end-up with your peptide solution at the desired and correct pH. You can easily test this yourself with a small volume of which you can measure the pH when you dilute for instance 10fold a 1mM NaOH (without your precious peptide of course) in your PBS. If the buffer pH remains unaltered, you can apply this strategy for your experiments.
The reason that you need a desalting step in case of DMSO is that there is no other way to get rid of that solvent. Despite DMSO being efficient in disrupting pre-existing aggregates (at least when slightly acidified, or in combination with HFIP treatment), it will interfere with cell-biology read-outs or with biophysical studies and with the behavior of the peptide in general.
I am sorry, what is the peptide you are using?
Isn't the Mr=4514.14 ??
If you have 0.1mg in 0.1ml of solution, you have a final concentration of 1g/L which corresponds to 0.2215 mM = 221.5 uM.
Isn't this correct???
@Hugo Vicente Miranda : sorry, i am clearing the concentration problem I have dissolved 0.1 mg of A-beta in 0.1 ml of HFIP after that it was removed by evaporation. after that i have dissolved it in 1 ml buffer so the concentration is 22 um, i am not measuring the od in hfip i am measuring when it is in buffer.
Hi there,
If you look at the peak at ~OD215 it is ~2.75. The Molar Extinction Coefficient for Amyloid-beta (1-42) at OD215 is 94,526 M-1cm-1 (Gunn et al., 2016). Assuming your pathlength is 1 cm and applying the Beer-Lambert Law, your Amyloid-beta (1-42) conc. should ~29.1 uM
I think this link is very useful,
http://www.instanano.com/characterization/theoretical/concentration-calculation-from-uv-vis-absorbance/
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