Hi, I'm having trouble creating a mass calibration when using slides coated in poly-L-lysine (for r-loop + protein interaction work). BSA, beta amylase, and thyroglobulin are sticking to the slides and I've had some success with beta gal. Are there any protein recommendations?
As soon as sample is added and mixed, molecules start landing on the glass slide. Variability in pipetting, mixing, and starting the recording will all contribute to variations in counts, even if starting with the same sample concentration. Most landing events occur right after adding sample and will decrease over the course of the measurement, so trying to keep an even pace (same number of pipette mixes up and down, same time between mixing, closing the lid and hitting record will all help keep your counts more reproducible between measurements.
Why am I seeing very high negative molecular mass histograms? Mass calibration seems ok and worked well for previous measurements.
A few times in recent experiments, I have noticed the first few readings have come up with negative mass values for my protein of interest (Events in ratiometric view appear as white dots). Eventually, using the same sample of protein and technique, the machine begins displaying a positive mass value and black event dots. Any thoughts as to why this occurs? I am sure to warm up the machine for 30 minutes before beginning. I have attached photos as to what the ratiometic view shows.
Another observation is that the negative mass values when this occurs are not reflections of the positive values. When the mass is negative, it is usually around -180 kDa, while when positive it is around 220 kDa.
As always, thank you for your time and help!
I am a new user of Mass Photometer. I was wondering that why do we see peaks around 0 and -ve molecular weights? Also, why do we see reflective peaks in negative ( they correspond to the molecular mass in positive). I would be thankful if anyone can explain this to me. Looking forward.
Hi Nicole,
Our technical support representative will get in touch with you.
Kind regards,
Team Refeyn