I am trying to measure the bond strength between streptavidin and biotin which was reported in literature to be around 150-250pN. In my case I have NHS-PEG-Biotin on mica sheet and streptavidin on the AFM tip. Both are covalently attached. I collect the force-distance curves at different loading rates and measure the amplitude or force required for bond ruptures. I plot a histogram for atleast 100 datapoints and fit a gaussian to find the mean rupture force. I am using MLCT cantilever from Bruker which has spring constant of 10pN/nm.
I have a representative Fx curve from my experiment and there are 3 peaks that can be seen.
I ignore the peak1 as it is due to adhesion and I consider peaks2,3 for my analysis as they are specific.
The issue is that I get a mean force of 40pN with one cantilever and 150pN with another cantilever at same loading rate.
I have the following questions:
I. Which value should be considered the correct one?
II. Why does this happen when the interaction between streptavidin and biotin is exactly same all the time?
III. What is the key to improve the reproducibility of experiment?
Any answers or opinions on my above three questions would be very helpful.
Hi
Once you analysed samples on FTIR then you can analyse the data using MATLAB software and can provide you all the related information for every peak or emission spectra.
One more thing for accuracy of the data in MATLAB take the Emission spectra of all the samples.
You can save data as pdf or word file and the Image of the emission spectra.
Hope this is helpful..
Hi Hamid,
I think you have misunderstood my question. It is regarding the force spectroscopy and not optical spectroscopy.
The force spectroscopy with AFM is a really sensitive technique, have you have consider the measurement of the cantilever constant for each experiment?, and for each cantilever?. Have you have consider the temperature difference for each experiment?. Any way, If you already had been fixed this variables you always can try again to make a reproducible experiment.
- Badges
- Science topic
- Similar topics
- Engineering
- Chemical Engineering