Effect of flash freezing of pelleted cells?

01 February 2017 1,646 3 View

Can MSCs be grown on a glass coverslip?

I am trying to grow mesenchymal stem cells on a glass coverslip (to be used for confocal microscopy). Does anyone have any experience with growing (not simply adhering) MSCs to glass coverslips?...

10 November 2014 7,159 4 View

What controls are needed in immunoprecipitation?

I have a custom antibody (it works for Western blot and immunohistochemistry) that I would like to try for immunoprecipitation. I know that the antibody successfully binds my beads (I'm...

02 March 2013 4,804 7 View

Chemical lysis of coelomocytes?

Does anyone know of a chemical lysis method for total protein isolation from coelomocytes? I'm currently using physical disruption in the presence of detergent. This gets me a fair amount of my...

31 December 2012 4,234 5 View

How can you evaluate EDU (labeling of newly synthesized DNA) in cells that are non adherent without flow cytometry?

We are studying cell proliferation in a cell type (from a marine invertebrate) that is non-adherent. Does anyone have suggestions for method(s) to evaluate EDU labeling in non adherent cells? We...

08 September 2012 3,714 17 View

Good tool for qRTPCR primer design?

I'm designing some primers for qRTPCR. Does anyone have any suggestions for a good qRTPCR primer design tool? (I expect to need to check for hairpins and dimers and what not, but would prefer...

08 September 2012 1,395 5 View

Suggestions for immunoprecipitation kits/protocols?

I'm somewhat new to IP and am looking for a good kit (or bead type) to isolate my proteins (large family of related proteins; we have an antibody specific for a conserved region). My antibody is a...

07 August 2012 5,033 5 View

How to evaluate EDU in cells that are non-adherent without flow cytometry?

We are studying cell proliferation in a cell type (from a marine invertebrate) that is non-adherent. Does anyone have suggestions for method(s) to evaluate EDU labeling in non adherent cells?...

07 August 2012 598 1 View

Has anyone tried using Bio-Rad Criterion XT gels with a home-made running buffer?

I'm looking to buy gels for the second dimension of IEF, and am attracted to the Bio-Rad Criterion XT gels - largely for their relatively long shelf-life (either the Bis-Tris with MOPS running...

07 August 2012 3,491 12 View

How can I quantify total protein concentration when a colored contaminant is present in my sample?

I am isolating a protein by metal affinity chromatography, concentrating and desalting it, and then drying it by vacuum centrifugation. I would like to be able to quantify my protein at various...

05 June 2012 9,987 61 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

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

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

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

Are protein samples in SDS compatible with ELISA?

I have used an AllPrep DNA/RNA/Protein Mini Kit QIAGEN kit to extract RNA and protein from my samples. At the end of the protein purification, I resuspend my protein in 5% SDS. Will these samples...

28 February 2021 7,370 3 View

Anyone that can help with Maxquant TMT16plex which is missing?

Hi I am very new to proteomics analysis. I have got raw files from core facility running thermofisher TMT 16plex 4 hours. I cannot find on maxquant the option TMT16Plex there is only up to 11...

25 February 2021 1,675 3 View

How to replicate the well conditions of a crystal hit with 20%(w/v) isopropanol?

I had a crystal hit in a well with 0.1M HEPEs pH 7.5, 10%(w/v) PEG 4000, and 20%(w/v) isopropanol. Why is isopropanol in w/v here if it's just solvent? How do I recreate this well condition?

25 February 2021 9,761 1 View

What's the concentration recommend for recombinant protein use in western blotting?

I'm currently working having problem with the concentration for recombinant protein use in western blot, the one been used is Recombinant Human CD19 Fc Chimera Protein-rndsystem which does not...

24 February 2021 8,514 3 View

Problem with ligating 2.8 kb insert into 8.5 kb plasmid?

I am trying to clone 2 copies of the same mammalian gene into the pSF-CMV-Ub-Puro Ascl (contains HindIII, KpnI and NheI cut sites) plasmid. This is what the final product is supposed to look like...

24 February 2021 6,310 3 View