Transcriptional or post-translational regulation?

I have came across a paper where they observed a delay timing of a drug inhibitory effect and insulin stimulatory effect respectively on purine synthesis, and thus, they suspected that the...

09 October 2018 9,919 0 View

How to process RNA-seq results?

I have obtained the RNA-seq results, which is in an excel file with all the fpkm values. May I know how do I check the gene expression profiles? Is there any software for this or I gotta manually...

04 May 2018 3,338 9 View

How to determine a protein is working downstream of another protein?

If overexpression of protein A can rescue the growth defect caused by mutation in protein B, can I consider protein A is working downstream of protein B? If yes, why is it so?

04 May 2017 7,522 24 View

Can anyone suggest method to extract membrane proteins?

Currently I am having problems in extracting membrane proteins (ammonium transporters), can anyone help? I used LDS in extraction and I did not heat the samples. Proteins in lane 1-4 seem like...

03 April 2017 773 20 View

Best temperature to dry DNA samples after ethanol precipitation?

I am preparing samples for BigDye sequencing. After PCR, I did ethanol precipitation to clean up the samples. In order to dry up the samples (after removing 70% ethanol), is it okay for me to dry...

09 October 2016 6,100 8 View

Overexpression of a gene is toxic to the cell? Why is it so?

Currently I am working on a new protein in fission yeast. Based on the predicted function, it is involved in transcription. We found that when this gene is overexpressed, the cell growth is...

06 July 2016 8,718 1 View

If one of the proteins in S.pombe was tagged with FLAG, is this pombe still considered as wild type?

One of the proteins in S.pombe that I am working on is tagged with FLAG, so can I still call this strain as wild type as it contains a fragment of foreign DNA? If no, what is the right term for it?

05 June 2016 8,096 3 View

Can I re-digest my digested PCR product?

I have digested my PCR product with NEB Not1 and Nde1 for 1hr. I have cut the band and purified the product. However I afraid the digestion is incomplete, can I re-digest them again? Is there any...

04 May 2016 2,655 17 View

Anyone has protocol for TaKaRa PrimeSTAR mutagenesis basal kit in English?

02 March 2016 4,844 6 View

What are the characteristics of a broad host range plasmid?

May I know what are the characteristics of a BHR plasmid? Is this kind of plasmid occurs naturally or we can try to engineer a narrow host range plasmid to become one?

01 February 2016 7,160 0 View

Why might my GFP tag be dissociating from my plasmid after transfection?

I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...

01 March 2021 9,310 4 View

Precipitate after adding laemmli buffer to protein solution containing Potassium Acetate?

Hi, I am running a size exclusion chromatography experiment with a buffer containing Potassium Acetate as a salt. I analyse these fractions through SDS-PAGE. After boiling my SEC fractions in...

01 March 2021 2,622 3 View

What is the most suitable server for predicting protein structures in bacteria?

I have used the i-Tasser several times, however it has been unavailable for several days. I tried the swiss-model, but the output was not very pleasant due to the model used. Is there any other...

28 February 2021 4,521 3 View

Do you need the CMV enhancer before the CMV promotor for a proper function of CMV on the gene that follows?

I'm a student and I have to produce a cell line with a knockin for the NRF2 gene with GFP. I have to put a promotor in front of the GFP because the gene will be too far away from the promotor of...

28 February 2021 7,127 2 View

Are protein samples in SDS compatible with ELISA?

I have used an AllPrep DNA/RNA/Protein Mini Kit QIAGEN kit to extract RNA and protein from my samples. At the end of the protein purification, I resuspend my protein in 5% SDS. Will these samples...

28 February 2021 7,370 3 View

Any specific pipeline or parameter to analyse total transcriptomics study of brain tissue samples?

I have two groups of brain samples, control and treated for example. It was total RNA nova seq sequencing. I tried all the available pipeline like: star+rsem+deseq2, Hista+stringtie+cuffdiff,...

27 February 2021 356 6 View

How to replicate the well conditions of a crystal hit with 20%(w/v) isopropanol?

I had a crystal hit in a well with 0.1M HEPEs pH 7.5, 10%(w/v) PEG 4000, and 20%(w/v) isopropanol. Why is isopropanol in w/v here if it's just solvent? How do I recreate this well condition?

25 February 2021 9,761 1 View

Problem with ligating 2.8 kb insert into 8.5 kb plasmid?

I am trying to clone 2 copies of the same mammalian gene into the pSF-CMV-Ub-Puro Ascl (contains HindIII, KpnI and NheI cut sites) plasmid. This is what the final product is supposed to look like...

24 February 2021 6,310 3 View

What urea buffer can i use for biotagged protein extraction from streptavidin beads?

Hi all, I have been doing research on biotagged LDB1 proteins in Mel cells. I purify the proteins using M280 streptavidin beads. The yield is very low so I've been trying to optimize it by...

24 February 2021 5,749 3 View

Can I use a protein-devoid condition as my negative control in ELISA?

Hello, I am running a sandwich-type ELISA where I am trying to inhibit a protein-protein interaction using small molecules. For each experiment, I determine my Z' (Z-prime) to determine the...

23 February 2021 7,429 1 View