I am measuring a sample of histone octamer which is in buffer containing 20mM Tris-HCl, 1mM EDTA, 2M KCl, 10mM DTT, 0.5mM Benzamidine and MilliQ water. I have prepared 1M Benzamidine in Ethanol and used this as stock solution for buffer preparation. I have blanked the Nanodrop 2000c Thermofisher Scientific using buffer without protein. Then I measured the spectrum (220 to 350nm) of buffer. The absorbance was not zero at all wavelengths, it is non uniform. Does it mean that blanking did not happen properly?
And then I measured the spectrum for histone octamers which are in assembly buffer and obtained negative values for wavelengths between 220-270nm. What does it say about the measurement or instrument?
How do I measure the right concentration of protein in this buffer?
Dear colleague!
I strongly recommend you to remove benzamidine since it include cyclic group which cn be more optically active than histone octamers proteins. if I understand correctly, you used benzamidine for proteases neutralization. Try to use other one and check their molecular structure (no ring structures).
Good luck,
Tatiana
Hi, Sumanth.
I recommend extract DNA again, dissolve in ddH2O for 18h and measure in the diluted stock with ddH2O again. Measure again.
Dear colleague!
I strongly recommend you to remove benzamidine since it include cyclic group which cn be more optically active than histone octamers proteins. if I understand correctly, you used benzamidine for proteases neutralization. Try to use other one and check their molecular structure (no ring structures).
Good luck,
Tatiana
Amended on 19 August 2018
I would like to express my opinion. Surely, Nanodrop is a deceitful tool. Nanodrop does not obey the "Lambert-Beer's Law". Direct photometry without purification is not obeying the "Lambert-Beer's Law" due to many contaminated molecules. HPLC-photometric detection uniquely obeys the "Lambert-Beer's Law", since purified molecule by HPLC is measured at the flow-through cell.
Please see Fig. 10 of the file "JCB Fucoidan transport", which has firstly indicated that the direct-utilization of photometer gives c.a. 3-fold lower values than the HPLC-photometric detection.
It is also noteworthy that HPLC-MS detection is not quantitative (personal communication via telephone from a kind engineer of Shimadzu Co., Kyoto, Japan).
Protein determination can uniquely give the true value via the quantitative and reliable HPLC-photometric method (please see file; HPLC-Surf-SEC protein determination method). Thus, I am repeatedly recommending the HPLC-photometric method for protein determination.
By the way, another quantitative protein determination method is our PDMD (Protein-Direct-Microsequencing-Deciphering) method. This unique method use famous Edman degradation reaction by covalently binding proteins to glass filters via EDC and by determining PTH-amino acids quantitatively with Shimadzu PPSQ microsequencer (please see file; HepG2 Fucoidan).
Dear Koy Hayakawa,
Thank you for responding to my queries.
According to the manual of Nanodrop, it used Lambert-Beer's law to measure the concentration of sample. In Pg # 8 out of 97, it says this.
I checked the instrument with various samples with known concentration and it works very well. I am facing issues only with this particular sample. I found that Benzamidine is not the reason by blanking the instrument with 1M Benzamidine in ethanol. There are no negative values in this case.
Does DTT have anything to do with this?
Thank you Dr. Cornelius.
I read that DTT is required to stabilize proteins.
Is there any alternative for DTT which will resolve the issue?
Hi Dr. Cornelius,
I understand that fresh DTT is in reduced form. And on interaction with protein it reduces the disulphide bonds of protein and gets oxidizes which is optically more active. So measuring the absorbance of fresh DTT will give zero value at 280nm.
I have histone octamers in a buffer with DTT which has gone through dialysis for 6.5hrs. Then they are flash frozen in liquid nitrogen. By now the DTT in the protein solution is already oxidized. So this will affect the absorbance readings.
What should be used as blank?
I cannot use the buffer with fresh DTT as this is in reduced form and the DTT in protein solution is in oxidized form.
Any suggestions would be helpful.
Thank you Dr. Cornelius.
Prior to posting the question on RG I tried blanking with the buffer that was used for dialysis and I was not careful about its storing temperature. That was the reason for getting such negative values.
Finally I understood the solution for this problem is to store the dialysis buffer protein-free solution in the same conditions as the protein-solution. So the protein concentration can be measured at any point of time without any confusion.
But do other labs do it in the same way?
It seems to be a complicated way.
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