Amulya Ramakrishna

5 Questions 4 Answers 0 Followers

Questions related from Amulya Ramakrishna

What are good alternative buffers to HEPES for DSS-based crosslinking studies of protein?

I am planning to perform crosslinking studies between two proteins and a peptide using DSS as the crosslinker. Both proteins are currently in SEC buffer, so I attempted buffer exchange into HEPES...

28 July 2025 202 2 View

What range of temperatures should I use in a gradient PCR to determine the optimal annealing temperature for PCR primers?

I have a pair of primers designed for a site-directed mutagenesis experiment. The Tm of the forward primer is 74.5 °C, and the Tm of the reverse primer is 70.5 °C. For amplification, I am using...

14 May 2025 5,908 4 View

What is the optimum concentration of NaCl required for the thrombin digestion of His tagged protein?

I'm working on a protein with a GST tag followed by a His tag. To remove the GST tag, I performed thrombin digestion at 4°C in the buffer containing 500 mM NaCl, 50 mM Tris (pH 8), and 5 mM...

01 May 2025 262 2 View

What is the optimum concentration of the Sodium citrate Dihydrate to be chosen for IEC cation exchange purification?

I am purifying a protein having pI of 6.63. So I am using Sodium citrate buffer (pH=5.5) for preparing wash buffer (Buffer A) and Elution buffer (Buffer B) for IEC. Kindly let me know what is the...

05 July 2024 2,780 2 View

Why is protein getting precipitated during dialysis?

I purified a protein containing His tag via Ni-NTA chromatography wherein the protein was eluted in elution buffer containing 500mM imidazole, 50 mM Tris, 500mM Nacl. Since my next step of...

01 April 2024 1,351 2 View