Do B lymphocytes respond to foreign DNA during transfections?

During transfection of B cells with plasmids/linear DNA fragments, do the large amounts of foreign (bacterial) DNA sequences activate signalling cascades, like that of TLR9 for CpG-rich sequences...

07 August 2018 2,230 1 View

Why is Lamin B1 not showing up in primary B cells' western blot?

06 July 2017 6,957 0 View

Peak shifts in cell proliferation assay using CFSE?

I am doing a time course experiment to study cell divisions. I have carried out the experiments in dark, given the light sensitivity of CFSE. I have analysed stained and unstained samples at every...

31 December 2016 7,763 11 View

What do the numbers after NP (nitrophenyl) mean eg., NP3, NP26, NP33, in the context of antibody affinities?

06 July 2016 6,382 1 View

How to perform nascent RNA quantitation?

Is it necessary to isolate nuclei from cells and use this RNA for qPCR? Or can specific primers be designed in intronic regions so that only unprocessed RNA will be picked, and use whole cell RNA...

05 June 2016 1,991 4 View

Why would a double-cut plasmid with ligase give colonies?

I am using a plasmid vector of about 8kb for cloning. After cutting with EcoRI and BamHI, I performed a ligation with T4 DNA ligase and transformed in DH5-alpha, and found that there were as many...

10 November 2015 8,216 11 View

Can the gene of interest be cloned after a viral 2A peptide?

I am interested in making a bicistronic system using viral 2A peptide to clone my protein of interest. The usual order of cloning is GOI-P2A-GFP (for example). Since the cleavage site is at the C...

07 August 2015 8,679 2 View

Why do I get a smear on the gel after the re-amplification of a PCR amplicon?

I have observed multiple times that if I use a PCR amplicon (with / without PCR cleanup) as a template for a subsequent PCR with the same primers, I get a smear on agarose gel. The size of the...

04 May 2015 9,447 20 View

How do I resuspend Trizol protein pellets?

I have been resuspending protein isolated by Trizol reagent in 1% SDS and storing at -20 degrees C. But everytime I need to use the protein for quantification, electrophoresis, etc., I need to...

04 May 2015 6,778 2 View

Ever transfected into M12.4.1 murine B cell lymphoma cell lines?

I have been trying transfections with this cell line, with hardly any success. Bicistronic IRES vectors don't seem to work Electroporation has been giving low efficiencies High concentration of...

01 February 2015 2,937 8 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

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

IHC on mouse liver hepatocytes?

Can anyone help me choose primary antibodies for mouse liver hepatocytes and also in conjunction Cd8 cells. Also Cd3 cells. I want to look at hepatocytes and adjacent immune cells in mice with...

01 March 2021 6,953 1 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

Which fluorescent dye to use for measuring total ROS production?

Is DCFDA appropriate for measuring total ROS production, or can it only detect certain types of ROS? If I want to measure total ROS production, which dye should I use?

01 March 2021 9,447 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

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

How to avoid ulcers in s.c. injected tumors ?

Recently, we have injected s.c. B16F10 tumor cells in BL/6 mice. At around 12-15th day some animals showed ulcerations in tumors , with a big hole in the middle (it looks like a volcano). We have...

01 March 2021 9,938 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