Leslie Spitalny

11 Questions 5 Answers 0 Followers

Questions related from Leslie Spitalny

Can alkyl diazirine fluorescent probes label BSA without light activation?

I am performing an in vitro in-gel labeling experiment by photo-activating a fluorescent alkyl diazirine probe with UV light in the presence of BSA to determine the required activation parameters...

08 September 2023 5,111 0 View

How long does it take L-homopropargylglycine to get cleared out of cells upon integration in nascent proteins?

I am planning to incorporate 1 mM L-homopropargylglycine into HeLa cells in methionine-free DMEM media. I know that the incorporation of this alkyne-containing amino acid into nascent proteins...

13 June 2023 3,920 2 View

How do you dissociate a 40 micron thick PFA fixed mouse brain slice?

I would like to use flow cytometry to analyze single cell expression of fluorescent expressed proteins. How do I dissociate a 40 micron thick, 24 hour PFA fixed mouse brain slice to achieve a...

11 August 2021 8,622 2 View

How do you reverse glyoxal fixation?

I am using a 3% glyoxal pH 4.5 solution to fix adherent cells. I was previously using 1% PFA and established the best way to quench the reactive aldehyde groups was with 1M Tris pH 8 for 1 minute...

10 August 2021 4,006 2 View

What is the best way to lift and dissociate fixed HeLa cells to keep them intact for flow cytometry analysis?

I am trying to lift, dissociate, and then flow fixed HeLa cells for fluorescence analysis. When doing this previously, the cells lift from the plate with 0.5% trypsin-EDTA treatment but they then...

30 July 2021 8,848 4 View

What is the best way to fix HCT116 cells to attain a uniform fixed membrane condition?

I am fixing HCT116 cells to use with a photo activation probe for cell membrane labeling. I have used 1% and 4% PFA in water as a fixative and fix on ice for 20 minutes. I then quench the reactive...

30 July 2021 4,870 0 View

What are the different rates of photobleaching for fluorescein vs rhodamines vs coumarins?

I am using confocal microscopy for photo-probe activation which requires photobleaching. The photobleaching rate of the fluorophores used for imaging will dictate the time of sample exposure. How...

16 June 2021 8,641 0 View

What is the best what to determine concentration of a fluorophore conjugated oligonucleotide?

Utilizing a aminoallyl-oligo of 33 base and a C12 linker, we are conjugating succinimidyl ester fluorophores together. After HPLC purification, what is the best way to quantify reaction yield and...

03 June 2021 2,555 3 View

What concentration of sample is required to measure the absorption spectrum on a UV/Vis Spectrophotometer?

I am trying to measure the absorption spectrum of a photo-caged fluorescent probe on an Agilent Cary 100 UV-Vis. I tried a 10mM solution in DMSO after a DMSO blank and the spectra had a broad flat...

11 March 2021 9,992 5 View

How to reduce cell surface reactivity of PFA fixed cells?

I am currently working on a project that involves photo-activation of fluorophore containing probes to spatiotemporally tag the surface of cultured cells or fixed tissue. There are two...

22 February 2021 8,073 1 View

What percent of serum should be added to cell culture media for use in scratch assays to analyze cell migration instead of cell proliferation?

I am using the scratch/wound healing assay as a platform to study cancer metastasis. This assay should mimic cancer cell migration for metastatic tumor initiation. However, after reading many...

27 August 2020 719 4 View