Furin western blot - 2 bands?

I am expressing furin gene in a furin deficient cell line (LoVo), but I always get 2 bands on my western blot ~ 87kDa, corresponding to furin expression and a lower band, of approximately 50kDa,...

08 September 2018 1,754 0 View

MRNA maturation after transfection?

I am having some problems trying to clone a mRNA, maybe due to it's amount compared to other mRNA. I have tried PCR using cDNA from oligo DT, specific or random primers, but with no success. I was...

07 August 2018 3,497 3 View

Transfection of S2 cells?

Does anyone work with expression in S2 cells? How efficient is the transfection using Lipofectamine 2000? Everytime I try to transfect using CaCl2 I get a cloudy media, and it seems that the...

06 July 2018 1,451 0 View

Contamination after hygromycin supplementation?

I am trying to establish a stable cell line after transfection with pcoHygro plasmid. Every time I supplement with the selection antibiotic (hygromycin) I get some small contamination (it seems...

06 July 2018 9,790 6 View

PCR using oligo dT?

Is it possible to perform a PCR, using as the reverse primer a oligo dT, the same used in the reverse trancription? I am trying to amplify a mRNA, but with no success

05 June 2018 6,831 4 View

Mouse vs rat?

I need to produce antibodies against my protein in 2 different animals, amd my university provide us mice and rats, but I am not sure if the secondary antibody will cross react between those...

01 February 2018 784 3 View

Baculovirus protein exporting pathway?

I am expressing a protein using Baculovirus / SF9 cells system, and I was wondering if, with the signal peptide for exporting the protein (pAcGP67a plasmid), the protein will go through Golgi...

09 October 2017 6,361 0 View

I am working with MinION sequencing technology, with a RNA imput. It generates several read that can not be assembled. Any clue how to analyze them?

The reads are not assembled as contigs, and I have tried some different softwares with no success, such as EPI2ME and Geneious. I have no idea how to align and check my sequences, I have tried...

08 September 2017 2,970 3 View

Can we classify HV and LV lines based on line length?

I have dataset which shows the length of power lines. I need to classify the lines based on the line length. Is there a rule to classify the High voltage (HV) and low voltage (LV) lines based on...

03 March 2021 4,116 4 View

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02 March 2021 3,060 3 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

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

What could be some reasons why unstimulated monocytes are getting activated?

Im doing PBMC isolation -> CD14+ enrichment using magnetic beads -> stimulation setup. My negative control is just cells in cRPMI but they seem to get activated over and over again.

28 February 2021 7,883 3 View

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28 February 2021 9,936 2 View

Has anyone had luck using cell lines other than HEK 293 (such as COS1) to package lentivirus?

I am looking to package lentivirus in endothelial cells but I am opposed to using 293 cells due to personal ethics regarding the source. I have done some reading and saw that COS1, COS7, & CHO...

25 February 2021 3,030 1 View