How to amplify 8.4 kb vector?

Hi all, I have been struggling to linearize an 8.4 kb vector by PCR for subsequent Takara Infusion cloning. I am currently using Phusion polymerase with the following protocol 98 C - 30 sec 98 C...

11 December 2019 6,126 4 View

Why is my protein expressed but not functional?

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...

31 December 2018 2,725 6 View

Problem with sfGFP expression in L929 cells?

Hi all, I have made an NLS-superfolding GFP (sfGFP) driven in pcdna3.0 and am trying to express it in L929 cells - mouse fibroblast line. Unfortunately, I am not having any luck. The construct...

04 May 2018 1,814 4 View

Amyloid Beta 42 Extraction?

Hi all, I am attempting to extract AB-42 from some of my 10 month old J20 mice for an Abeta ELISA. I initially perfuse the mouse with heparinzed PBS then snap-freeze one hemisphere on dry ice....

01 February 2018 582 1 View

RNA Isolation Troubleshooting

Hi all, I am having some issues with my Trizol-based RNA isolations. I am attempting to harvest RNA from bone marrow derived macrophages (3x10^6). After the initial separation with chloroform I...

07 August 2017 9,554 11 View

High Cq values for GAPDH control?

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...

07 August 2017 4,926 4 View

Thermo Fisher cAMP ELISA no signal?

Hi all, I am using the cyclic AMP competitive ELISA kit from Thermo fisher and have been not getting a signal from either my standards or samples. I observe the green color change after adding the...

07 August 2017 5,266 2 View

How to traffic cytosolic protein to membrane?

03 April 2017 1,301 4 View

Error-prone PCR Troubleshooting?

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...

31 December 2016 411 3 View

Fusion Protein Degradation?

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...

10 November 2016 5,033 13 View

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02 March 2021 3,060 3 View

New Journal, "Integrative Psychological and Behavioral Science" -- do you not know, and have you not seen, this done before?

Question to you and THEM, the New Journal, "Integrative Psychological and Behavioral Science" -- do you not know, and have you not seen, this done before? There appears to be a core problem for...

02 March 2021 3,024 2 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

Could you please recommend some good journal that accept very long revie papers (approximately 6000+ words) in neurosurgery, cancer biology ?

Could you please recommend some good journal that accept very long revie papers (approximately 6000+ words) in neurosurgery, cancer biology ?

01 March 2021 8,087 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

Where to find data on trees in tropical agroforestry?

Hi, I'm looking for data (mainly related to management: growth rate, canopy size, soil and climate preferences, etc.) about tropical trees used in tropical agroforestry. Have you ever heard about...

28 February 2021 7,356 8 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