21 Questions 53 Answers 0 Followers
Questions related from Lyra Chang
Hi All, I have used MBP tag to help solubilize my target protein. It works really well. However, whenever I cleave off the His-MBP tag and try to clean up the sample by passing through another Ni...
03 March 2019 1,029 6 View
Hi All, I am trying to establish a good DNA degradase protocol for characterizing the global DNA methylation level of mammalian cell genomic DNA via LC-MS. I uses the DNA degradase plus from...
06 June 2018 2,891 12 View
Hi All, Could anyone suggest a economic & good stereo zoom microscope that can allow me to inspect & manipulate protein crystals (with the cryoloops)? We don't need polarized light. As...
03 March 2018 3,079 1 View
Hi All, I am just curious about how HeLa cell contaminated so many cell lines? I mean, if we use disposable pipettes, disposable aspirating pipettes, disposable tips, HeLa cell shouldn't be...
02 February 2018 604 5 View
Hi All, I want to select a cell line for our drug study but it has to be highly sensitive to p53 induction - i.e. overexpression of p53 can easily kill the cell line. Any suggestion will be...
10 October 2017 1,260 3 View
Hi All, I am starting to develop a LC-MS/MS based quantification method to determine the global DNA methylation level in cultured cells after compound treatment. I am following this paper right...
06 June 2017 6,090 3 View
Hi All, I am developing a high throughput fluorescent polarization assay against my target. It is a histone binding domain and the fluorescent probe is attached to a peptide. The protein domain is...
03 March 2017 8,515 26 View
Hi All, I was told that it is best practice to attempt growing protein crystals only after gel filtration column, but my protein is already quite pure after ion exchange column. I wonder have...
01 January 2017 9,533 6 View
Hi All, We have problem getting good transfection of PX459 by Lipofectamine 3000 into A549 cells (I am not 100% sure, just that we don't get any editing with all 4 gRNA we designed based on...
08 August 2016 7,594 9 View
Hi All, I am trying to test how my mutated protein affect the methylation of repeat sequences in genome (ex. LINE-1 and Alu). However, I found it quite hard to get the full length sequence of...
08 August 2016 5,873 2 View
Hi All, I have done tons of MTT assay lately for my project and have so many plates filled with the purple formazan solution (I used the SDS lysis method so no DMSO present). I just have no idea...
08 August 2016 5,898 3 View
Hi All, Our lab will have some money to buy equipment pretty soon and we are interested in buying a liquid nitrogen mammalian cell line storage chamber/freezer. For me, taking out frozen cells...
04 April 2016 9,404 7 View
Hi All, I am currently starting to develop a high throughput assay against my target which is a Ring E3 ligase. Does anyone have any successful experiences to share? Also, any input on good...
04 April 2016 9,964 2 View
Hi All, Since our lab doesn't have BL2 tissue culture room, I used other lab's space for generating Lentivirus and infecting the cells with virus. Now I am about to start my Puromycin selection. I...
03 March 2016 5,846 2 View
Hi All, I am currently starting to think about some future independent research projects that related to virology. I know which general aspect I want to study about virus; however, I can't decide...
02 February 2016 6,076 6 View
Hi All, Have anyone try KO a gene from mammalian cell line with Cas9 protein + vectors that expressing gRNA? Also, for generating KO clones, is it better to use protein-based or DNA-based CRISPR...
12 December 2015 845 6 View
Hi All, I am planning to use 2gRNA & wild-type Cas9 to KO my target protein. I plan not to use HDR for my purpose but hope that by using 2gRNA I can create a specific deletion that help me...
12 December 2015 2,808 7 View
Hi All, I am planning to use 2 gRNAs and wt-CRISPR to KO my target protein from the cell lines I pick (ex. Hepatocellular carcinoma cell lines). I want to clone both gRNAs into vectors and...
12 December 2015 7,771 5 View
Hi All, Our lab is trying to use AlphaScreen to find protein-protein interaction inhibitors for a nuclear protein (90kDa). We thought it would be easy but our design didn't work (no binding...
05 May 2015 2,033 5 View
Hi All, I am trying to purify a recombinant His-tag protein from insect cell (baculovirus expression system) and my protein is a nucleus protein. Previously during clone selection stage, I used...
05 May 2015 6,159 10 View
Hi All, I am currently purifying a in vivo biotinylated protein from E. coli. It has a C-terminal Avitag (to allow in vivo biotinylation). I found that when I switch the buffer to lower salt...
01 January 2015 5,579 4 View
