How to prevent MBP protein associate with protein of interest after TEV protease cleavage?

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...

02 March 2019 894 6 View

Does anyone has good DNA degradase protocol (DNA to nucleoside)?

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...

05 June 2018 2,829 12 View

Cheap & good stereo zoom microscope for manipulating protein crystals?

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...

02 March 2018 3,019 1 View

How did HeLa cells contaminated so many cell lines?

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...

01 February 2018 529 5 View

Can anyone suggest cancer cell lines that are highly sensitive to p53 overexpression?

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...

09 October 2017 1,202 3 View

Good protocol for global DNA methylation measurement by LC-MS/MS method?

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...

05 June 2017 6,027 3 View

Fluorescent polarization - strong fluorescent increase after adding protein?

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...

02 March 2017 8,446 26 View

Have anyone try growing protein crystals without gel filtration step?

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...

31 December 2016 9,469 6 View

How can I clean the waste generated from MTT assay?

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...

07 August 2016 5,805 3 View

Transfection reagent for PX459 (CRISPR-Cas9 gRNA vector)?

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...

07 August 2016 7,433 9 View

How can I measure MMP-1 protein activity or protein level from liver tissue?

Instead of checking only the mRNA level, I want to check the active protein level of MMP-1 in Liver tissue from mice. How can I do that?

03 March 2021 1,763 2 View

Can linear DNA be used as template for site-directed mutagenesis?

Is it possible to induce site-directed substitution mutation by quick-change method on linear dsDNA? or it has to be cloned in some vector? If yes, should it be treated with the Dpn1 enzyme...

03 March 2021 401 4 View

Does any one know how i can prepare liver tissue with transmission electron microscope with good fixation?

i have problem in preparation of liver tissue with bad cell organelles and how i can adjust osmolarity

03 March 2021 8,890 1 View

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02 March 2021 3,060 3 View

Does anyone have a recommendation for yeast reporter gene plasmid/protocol?

Hi, could anyone recommend a plasmid and/or protocol for reporter gene assay in S. cerevisiae? I want to assess the effect of growth conditions on a transcription factor, so I want to clone it`s...

01 March 2021 210 1 View

Issues with Permeabilization during Flow Cytometry?

Hello Everyone. Currently I am working to characterize macrophages in the myocardium after ischemia-reperfusion injury in rats. Due to the low total cell number isolated from rat hearts I can...

01 March 2021 3,867 3 View

Which cell line should we use to perform biological dosimetry experiments?

We are preparing some experiments based on irradiating cells under different conditions in order to evaluate the effects in terms of DNA damage, genetic expression, etc. As our project is...

01 March 2021 3,355 3 View

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

01 March 2021 3,607 3 View

Is CD44 degraded in lysosome together with hyaluronan after CD44 mediated endocytosis of hyaluronic acid?

Also when RHAMM binds hyaluronic acid, they get internalized, will RHAMM also be degraded? Or both CD44 and RHAMM will be transferred back to the cell membrane? Asking for breast cancer cell line...

01 March 2021 8,169 2 View

Expression cloning by using cDNA,do I need to modify the cDNA first?

I am going to have a expression cloning of mammalian gene by using shuttle plasmid to transforming the E.coli However I don't know I should only inserting the Coding sequence ,or I can...

28 February 2021 5,440 3 View