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Questions related from Sushanth Kumar
Hi, I have an image below of microglia (green) surrounding various beta-amyloid plaques (red). What I would like to do in ImageJ is to determine the % of the plaque perimeter that is covered by...
21 October 2019 7,816 3 View
Hi guys, I am over-expressing the protein MLKL along with various tagged forms in an MLKL KO cell line. Despite being able to being able to visualize the protein by both immunofluorescence...
17 January 2019 6,615 5 View
Hi all, I would like to generate amyloid beta oligomers for treatment on glial cells to assess cytokine production. I have come across a number of protocols but have been unsuccessful in...
11 September 2018 4,027 0 View
Hi all, I am studying necroptosis in BMDMs. After an overnight stimulation I need to stain with propidium iodide to examine cell viability. I would like to also counterstain with DAPI to get an...
28 August 2017 6,413 4 View
Hi all, I have some BMDMs from various genotypes which I have stimulated overnight with LPS+zvad and LPS+zvad+Necrostatin to study differences in necroptosis. Most protocols I have read have used...
17 August 2017 7,710 3 View
Hi all, I am performing qPCR on Trizol extracted RNA from BMDMs stimulated with LPS. I am probing for TNF-alpha cytokine production as well as a few other candidates. My TNF qPCR results yielded...
09 August 2017 5,672 3 View
I have a cytosolic protein that I would like to have traffic to the plasma membrane. The protein should not tether to the membrane but be localized to it. I am working with HEK293 cells. 1. There...
02 April 2017 3,636 3 View
Hello, I am using TriLink's Mutagenesis dNTP mix to perform EPR on a 1.3 kb fragment. I have been unable to observe a PCR product when following their recommended cycling parameters. I am able to...
19 January 2017 140 3 View
Hey guys, I have noticed that one day following passaging that instead of distributing evenly across my 10 cm plate that there are large areas that are largely devoid of cells. There are cells in...
13 December 2016 5,626 1 View
Hi all, I have an insert that I am attempting to ligate into pcDNA3.0 cut with HindIII and KpnI. I get a ton of background colonies even after dephosphorylating the vector (I know my phosphatase...
23 November 2016 1,009 4 View
I have generated an mCherry fusion protein construct in pCDNA3.0. The construct expresses in HEK293T cells as verifed by mCherry fluorescence and immunoblotting against mCherry. When I run my...
02 November 2016 8,847 12 View
I am performing a study in which I am observing the interaction between two proteins following a stimulation. Each protein is tagged with half of a GFP moiety and therefore upon interaction would...
28 October 2016 8,497 1 View
I have noticed in several constructs that people will include a triple polyA cassette (typically SV40). Is there a significant improvement in message stability with 3 poly A sequences as opposed...
07 October 2016 9,863 1 View
I have a 4.8 kb insert that I have been attempting to subclone into pcdna3.0 for months now. In pUC57 the insert seems to propagate just fine and maxipreps give good yields. The following day...
30 August 2016 495 3 View
I would like to subclone in a 25 amino acid group into various intracellular domains of a GPCR that I am studying. I will be making about 8-10 constructs and I will screen for functionality using...
31 July 2016 7,491 3 View
Hey all, I am attempting to transiently transfect three plasmids into CHO cells. My plasmids are a CRE-luciferase (minimal promoter), pRL-Tk-Renilla, and my gene of interest driven by CMV. On my...
20 June 2016 3,688 2 View
