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Do you have any siRNA Nucleofection® results using concentrations lower than 50nM?

Yes. Please click on the attached Technical Reference Guide: "Designing an RNAi Experiment Using Nucleofection®". On page 3 you can find a table with some examples.

What antibodies can be used against the GFP produced by pmaxGFP and pmaxFP-Green vectors? What antibodies can be used to detect denatured GFP? What antibodies can be used to detect the RFP expressed by pmaxFP-Red?

Most of the commercial antibodies for GFP do not detect GFP expressed from pmaxGFP or pmaxFP-Green vectors. The origin of the protein expressed by the pmaxGFP control plasmid and pmaxFP-Green is from the green fluorescent protein CopGFP, cloned from...

For hepatocytes, is plating density a concern post Nucleofection®?

Yes, the optimized protocols for the standard Nucleofector® recommend the use of 6 well plates. Plating into a 12-well plate will pose a problem as less cells will attach at high densities.

Four hours after Nucleofection®, I can see the hepatocytes have attached to the well, but the morphology does not look correct, should I be concerned?

There is no reason for alarm. The hepatocytes may not exhibit normal morphology a few hours after Nucleofection® but by 24 hours post Nucleofection®, morphology should be normal. Remember to perform a fluid change about 4 hours post...

I want to stimulate human primary T-cells with anti-CD3 and anti-CD28. Do you have a detailed protocol for this?

Yes, the detailed protocol is part of our 4D-Nucleofector® Protocol for stimulated Human T Cells.For optimal stimulation of the transfected cells we recommend coating 96-wells plates for stimulation [Nunc Immuno™ Plate C96 Maxi Sorp™, Cat. No. 430...

Which reporter gene should I choose for my reporter gene experiment?

In general, all reporter genes can be used.For some applications (e.g. reporter gene experiments in various primary cells, experiments with late analysis time point due to starving and /or stimulation) a reporter gene with a longer half life than...

What is the amount of DNA I can use in my Nucleofection® Experiment?

This totally depends on the Nucleofector® system you are working with. While in the 100 µL reaction vessels usually 1 - 5 µg DNA is being used, for the 20 µL reaction vessels (e.g. X Unit, 96-well Unit and HT...

Is it more difficult to detect DsRed by FACS than GFP?

Yes, DsRed is much less bright than GFP and is more difficult to detect by FACS and microscopy because it bleaches quickly.

When should I begin looking for the Luciferase signal after Nucleofection®?

The time course of plasmid expression after Nucleofection® might be different from the time course seen with other transfection methods. We recommend looking at different time points and start with the first time point as early as 4 to 6 hours post...

What should I pay attention to when transferring my reporter gene set up from a cell line to a primary cell?

In general the overall expression level differs between different cell types. Don't expect the same level of gene induction when working with primary cells. You might need to change to other DNA amounts or ratios.
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