what are the acceptable ct (threshold cycle) values for amplification (qRT-PCR) of a plasma miRNA?

We are optimizing protocols for plasma miRNA isolation and amplification. However for some miRNA, Ct Values fall in the range of 37-40. Negatives are clean. Should we ignore such low Cts as...

06 July 2018 3,618 7 View

Can anyone give advice on ShRNA transfected stable clones?

We have been using shRNA plasmids to silence the expression of an endogenous gene. We have realized that some of the stably transfected clones grow in the presence of selection marker containing...

07 August 2014 8,345 7 View

Problems in pelleting of biotinylated cells.

We biotinylate live cells (adherent monolayer of Ishikawa) for 1 hr at 37C. This is followed by quenching and washing with PBS. However after scrapping in PBS, followed by centrifugation at 2000...

31 December 2012 1,344 20 View

How reliable is the chemiluminescent detection of proteins (western blot) for relative quantification?

Our experience tells us that the intensity of immunoreactive bands on luminograms, after chemiluminescent ECL advanced detection does not always correlate with the protein load.

10 November 2012 2,356 7 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

Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence?

I am on the lookout for the Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence. Does anyone know where I can find it? Thank you in advance

03 March 2021 3,568 1 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

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

Why does two different bioinformatics tools for the same proteins show different subcellular localizations?

During a computational analysis, I found that for a protein of a plant, two different subcellular localizations are seen using CELLO and WoLF PSORT. For example, in CELLO, it identified the...

01 March 2021 6,729 2 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

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

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