I have mostly done computational works and the transition to experimental work is slightly demotivating as I am stuck at each stage starting from whether to wear gloves for certain things to why my experiments are not reproducible!
At this stage, I am seeking an answer to how can I weigh my peptide correctly to make sure I am getting the same concentration as I planned?
For example, I want to get 1mM (just an example) concentration for my peptide and I calculated the required mass to be 0.7134 mg. Now a few questions which bother me are:
1. Since the weighing balance can only allow two decimal places, so should I round it off to 0.71 (because 3 is less than 5) or should I round it off to 0.72 (because it is likely that I will lose some peptides anyway while weighing!)
2. Even though I know theoretically the concentration of my solution, should I still measure maybe using nanodrop or some other way? (and how accurate it will be if no aromatic aa)
3. Is there anything else I should be taking care of here?
Also, if there is any authentic website or paper to read about this basics, please recommend.
Thank you all in advance.
You need to calculate the concentration based on actual weighing done on balance (may be 2 or more decimals as per balance capacity).
Do not calculate concentration first followed by required weights as you will not get same weight on the scale due to various reasons (manual/system errors).
If you mean the Mt. Weight by using amino acid analysis and each amino acid weight 120 after lost the water molecular (18).
Hi Chandni!
If I understood well, you need to weight an amount of your peptide(s) that is just too small for the accuracy of your balance.
What I would do in your case is to weight an amount of your peptide ten times higher, 7.13 mg instead of 0.71 mg, and go for dilutions. So, I would weight 7.13 mg, which should be enough for an accurate measurement, and dilute it in, for instance, 100 uL (Stock 0). I would take 10 uL of those 100 uL (so you are actually taking 0.713 mg of peptide) and add them to the required amount of your buffer/solution to get the concentration you need. You can adapt all the numbers here to your own convenience, of course!
Also, I would suggest that for this first solution (Stock 0) you use milliQ water, so you can just lyophilize the rest of your peptide without purifying it again.
I hope it helps and do not hesitate to ask if you have further questions! =)
Hi,
Please check this
https://www.blueskypeptide.com/blog/properly-weigh-peptides/
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