19 Questions 11 Answers 0 Followers
Questions related from Fatin Safiudin
Hi! So i attempted to understand a novel protein behavior towards heat application by analyzing its secondary structure change. I subjected the protein to a thermal denaturation analysis using...
07 August 2024 1,929 3 View
Hi! Recently I attempted a two step protein purification (affinity- GSTrap FF resin then iex- HiTrap Q FF) for my recombinant protein. Capturing step (affinity - GSTrap FF resin) I connected two...
01 December 2023 5,077 0 View
Hi. Recently, as a mean to increase yield, I added 0.1% of Tx-100 into all of my IEX purification buffer including the sample injected. However, when analyzed on SDS -PAGE, I observed that there...
28 November 2023 2,281 3 View
Hi, I am purifying a synthesized GST tagged membrane protein with the following condition: Crude preparation: 100ml culture was centrifuged at 12000xg for 15 min The pellet was resuspended with...
10 August 2023 4,115 2 View
We found an old dry packaged dialysis tubing in the lab and plan to use them for buffer exchange prior to ion exchange chromatography. It was kept in room temperature with no visible sign of...
09 August 2023 6,875 4 View
Hi, I was conducting few physochemical analysis for my target protein and found out that the protein located in the transmembrane region. Usually, transmembrane protein are hydrophobic and will...
04 August 2023 4,241 2 View
I keep all my partially purified protein in the respective elution buffer (20mM NaP, 0.5M salt, 100mM imidazole) at -20° freezer. I plan to do a buffer change today for my next polishing step....
07 July 2023 4,976 2 View
Should I take into account the protein tag (His/ GST) during the calculation of pI to choose which ion exchanger I should use? Or should I consider only the real target protein sequence only?
05 July 2023 1,463 2 View
My N -terminal GST tagged protein fail to purify using this condition: 30ml sample (3mg of crude protein) injected at flowrate (0.5ml/min), 40ml wash (PBS pH 7.3) and 5ml elution (50mM Tris HCl +...
06 June 2023 5,708 4 View
I performed zymogram analysis for lipase using this protocol: 1. Run Native -PAGE until adequate migration (roughly 80V for top gel: 30min, 120V for bottom gel: 2 hour) at 4 °C. Cathode connects...
06 June 2023 6,608 5 View
My study is on lipase and I planned to used p-NP palmitate method as the activity assay. The recipe includes Tris HCl buffer pH 9 + Triton X-100 + isopropanol. I stirred the mixture with heat then...
22 March 2023 3,275 4 View
I was looking for any recommendation for protein purification system for my research. We dont have much budget for akta and are looking for affordable alternative. We do have HPLC in the lab, can...
08 March 2023 7,559 3 View
I conducted gravitational His tag protein purification manually and observed that the A280 of my crude is similar to the eluent post sample application. How is this possible? Does the His protein...
30 November 2022 1,625 5 View
Ive made a mistake of preparing 0.1M NiSO4 in DURAN glass bottle. Can I reuse the bottle? If so, how can I neutralize the bottle from any traces of NiSO4?
17 October 2022 1,866 2 View
Hi, I couldnt find anywhere the exact percentage or amount of cysteine need to be present for my protein to be considered as cysteine rich. May I know the exact number/ percentage? for example >1%
25 August 2022 8,252 2 View
I did an in silico analysis for my protein to understand its secondary structure by using two servers; PSIPRED Workbench (http://bioinf.cs.ucl.ac.uk/psipred/) and SOPMA...
22 August 2022 417 3 View
Hi, recently I tried to express my genes (from plant) into Rosetta De3 pLysS using this protocol: 1. 1% overnight culture into a new media + antibiotic, incubated to grow until OD600= 0.5 2....
11 August 2022 2,578 1 View
My gel set can hold 4 gels but my samples can fit in 2. I have only one dummy plate so I have one extra empty slot. Can I use an empty SDS gel as dummy? And can I reuse the gel back with sample?
02 June 2022 1,830 2 View
Hi, Ive prepared my ligation mix for sticky end ligation in the ratio of 1:3, 1:6 and 1:9 calculated using NEBio Calculator with NEB T7 DNA Ligase at 25°C for 20 minutes then transformed them in...
09 January 2022 999 2 View
