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Questions related from Jian Wang
Hi all, I have many stable cell clones that originated from single cells. However, I found (by IF) that not every cell expresses the protein, and this percentage varies between clones. I was...
04 May 2023 5,694 3 View
Hello. I wanted to prepare whole cell extract for western blotting. Currently, I Step 1. Lyse cells in detergent (e.g., TritonX-100), Step 2. Take some lysate for determining total protein...
18 April 2023 6,415 15 View
Hello the community. I had an interesting observation recently: I trypsinized HEK293T cells in a 12-well plate and afterwards, rather than adding complete medium, I added PBS to the cells. I...
08 March 2023 661 7 View
Hello all! I am new to cell culture and just recently started to use HEK293T cells (adherent). Sometimes when I passage cells I will need to make sure all cells in the suspension are ideally...
30 April 2022 7,421 16 View
Hi all, just a small question. I run DNA gel using NEB 6x DNA loading dye as the loading dye and Gel red as DNA stain. Sometimes when I have very heavy DNA bands (e.g., after PCR) I can even see...
24 December 2021 2,182 9 View
Hi all, I have a protein which, once purified (e.g., in its elution buffer), is very easy to aggregate, as manifested by the formation of flocculent mass floating in the solution, and a reduction...
04 August 2021 9,142 8 View
Hi everyone. I am currently using Invitrogen pCR-Blunt II-TOPO kit to ligate my PCR fragments into the TOPO vector. However, most of the time I obtained very few E.coli colonies (fewer than 3 on...
10 July 2020 1,422 3 View
Hi everyone. Just out of curiosity. I am using High Five insect cell as the protein expression system. Cells are resuspended in 50mM Tris-HCl (pH 8.4), 200mM NaCl, 5% glycerol. After cell...
10 July 2020 6,922 6 View
Hello! I perform protein purification on a weekly basis and I have been really struggling with some proteins in terms of concentrating them. For all proteins I use centrifugal concentrations...
01 January 1970 2,509 11 View
Hi all, I have many stable cell clones and I have found that (by IF) only a subpopulation of cells express the protein of interest (POI) in each clone. This is how I generated the clones: 1. I...
01 January 1970 9,936 0 View
