Jian Wang

10 Questions 31 Answers 0 Followers

Questions related from Jian Wang

How common is gene silencing in stable cell clones?

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

How to prepare whole cell extract SDS-PAGE samples?

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 6,416 15 View

Why does Trypsin + PBS cause cell coagulation?

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 662 7 View

How to make single HEK293T cell suspension?

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 7,422 16 View

Why can I sometimes see DNA bands with naked eyes on agarose gel?

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 2,183 9 View

Why do some purified proteins tend to aggregate even by mild agitation?

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 9,144 8 View

TOPO cloning fails to integrate my PCR product?

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 1,425 3 View

White stuff found on the surface of insect cell supernatant?

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 6,923 6 View

Some proteins take forever to concentrate ...

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 2,510 11 View

Only a subpopulation of cells in a stable clone express the protein of interest.

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 9,937 0 View