🧪Thermal Denaturation of Proteins using Circular Dichroism || Practical Biochemistry
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- Опубліковано 23 жов 2024
- This is a great technique to get Tm (melting temperature) to compare stabilites and denaturation kinetic
If you have any questions or video suggestions please don't hesitate to leave a comment 😊
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I love your videos
Good informative tanks,👍🌹
Good keep up
Can you do a video on ANS fluorescence spectroscopy
Hi! Great videos! Could you write the reference number of your couvette? I use a foldable couvette 0,1mm but I have problems with evaporation and bubbles, especially when the probe is heated. Also do you have any tips for different results of the same sample? Sometimes I put on the same protein (from the same eppendorf), the same amount and I think that the results are different. Maybe its because the couvette that enhances evaporation? Thanks :D
which is best book to study and learn about these techniques?
As a graduate student it's been a long time since I had an actual text book to read. I usually read many journal papers and manuals to construct an understanding of a technique.
Hi. How can you analyze the CD spectra for the secondary structure of the protein? My protein change the CD spectrum pattern in the present of EDTA.
What changes in CD spectrum do you see?
What is the role of Liquid Nitrogen in that case??
It's for the main lamp, otherwise it will get damaged
Hi, have you ever worked with protein aggregates using circular dichroism? I solubilize my protein, did some dialysis experiments and used CD but I'm having a hard time interpreting these results
Hello. What kind of aggregates are they? Amorphous or like fibrils, and how would you describe the data you are getting? Are you getting a lot of noise on the left side of the curve?
@@biochemistmelo Thank you so much for responding! This is awesome! They are amorphous and the graph starts at zero (right side) and then keeps going down (-1 mdeg) from 230-215nm (-2 mdeg) at 210 and goes up (-1mdeg) and the 205 nm goes down (-4 mdeg). Yes! It looks like a lot of noise on the left side of the curve as you mention!
@@nicholemontero8481 can I also ask what you dialyzed the samples in? And concentration of sample
@@biochemistmelo Of course, I used 20 mM NaH2PO4, 0.5 M NaCl! sample was a little low 3.56 µM
@@nicholemontero8481 yes both the NaCl concentration is really high and the protein is low (try 5x more). I would reduce the salt as much as possible if it doesn't affect your protein.
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How can you analyse the data to determine the exact Tm and measure the cooperativity?
So there is a baseline for when the protein is still folded and when its completely denatured with a slope in between. The midpoint of the slope is Tm. For example if it starts unfolding at 30 and finishes at 50, the Tm is 40.
The cooperativity comes from how steep the slope is. If you have something that's almost a sharp 90 degrees and another that's 30 deg, then the sharp transition is the more cooperative unfolding. If you have more than 2 datasets then measuring the slope (difference in y/x). I suggest starting at a really low temp like 10 and ending at a high temp like 80 so you can clearly see the baseline before and after the main slope of transition.