Sam Siljee

19 Questions 71 Answers 0 Followers

Questions related from Sam Siljee

Can you correct fold-change in TMT proteomics experiments?

Hi all! I've run both LFQ and TMT 18-plex proteomics on the same protein extracts. My experiment consists of two study conditions, and 8 biological replicates. After digesting my protein...

20 January 2025 2,520 2 View

What is the best way to photobleach eGFP?

Dear all, I'm doing cell culture with lentiviruses including eGFP. While this is a very useful way to check transduction efficiency, it does take up a channel with subsequent immunofluorescence...

21 February 2024 1,715 2 View

What is the best far-red dye for fixed cells?

Dear all, We have a Olympus FV1200 confocal microscope with 405, 473, 559, and 635 lasers. I'm looking for a nuclear dye (fixed cells) in the far red range, does anyone have any recommendations...

24 January 2024 6,554 4 View

Cell signalling - how is specificity maintained through common intermediates?

Dear all, I have a basic biology question, but have yet to come across a satisfactory answer. Many signalling cascades downstream of specific receptors occurs through intermediates such cAMP,...

29 October 2020 210 4 View

Basic question - how to make a cell dissociation buffer?

Dear All, I'm looking to make a cell dissociation buffer from lyophilised collagen I (Gibco, 12648010). The protocols I've been looking at all specify concentrations of enzymes in terms of...

07 October 2020 768 3 View

What is a suitable inmmunohistochemical tissue negative control for (pro)renin receptor?

Dear All, We will be performing immunohistochemical staining for (pro)renin receptor (ATP6AP2), and I would like to perform a tissue negative control along with the standard isotype control. I'm...

02 July 2020 3,194 0 View

Expected phosphopeptide numbers from primary lung basal cells

Hi all, I have recently done a pilot protein extraction from primary lung basal cells with phosphopeptide enrichment using the protocol described by Humphrey et. al, and found 2433...

01 January 1970 1,253 6 View

Distribution of club cells in airway epithelium

Dear all, I am growing human primary airway epithelial cells from excised normal lung tissue in 2D ALI culture. In order to validate my model, I am doing immunofluorescence staining of the...

01 January 1970 2,979 0 View

Artefact in volcano plot

Hi all, I've noticed a suspicious pattern in some volcano-plots I've made of proteomics data. Specifically, I've noticed points lining up, seeming to show a direct mathematical relationship...

01 January 1970 3,681 4 View

Bridging channels and TMT in mass spectrometry for proteomics

Dear all, I'm working on the finer details of my experimental design, and have some questions regarding bridging channels for TMT based experiments. I have two conditions to test, across nine...

01 January 1970 8,947 1 View

Over-representated vs Upregulated

Hi all, I'm busy building a shiny analysis pipeline to analyse protemics data from mass spectrometry, and I was wondering what the exact difference is between the terms Over-represented, and...

01 January 1970 4,205 3 View

GO term enrichment analysis for proteomics in R

Dear all, I'm building a shiny application for analysis of mass-spectrometry based proteomics data (for non-coders), and am working on the section for GO term enrichment. There are a wide variety...

01 January 1970 4,867 5 View

Cryopreservation media for primary lung basal cells

Hi all, I'm growing primary basal cells from lung digests, and was wondering if anyone has recommendations for cell freezing media for primary lung basal cells. Previously I have been using...

01 January 1970 6,454 8 View

Cryopreservation of lentivirus vectors

Dear all, I'm using lentiviruses to knock down p53, and have had remarkable success so far with un-concentrated harvested media. I have noticed that the transduction efficiency declines over the...

01 January 1970 4,483 3 View

Batch to batch variability with DIA acquisition in mass spectrometry

Dear all, I'm starting out with mass spectrometetry, and am thinking of using DIA for improved quantitation of low abundance proteins, and to save on buying TMT reagents. Does anyone know how the...

01 January 1970 2,959 6 View

Cell sorting from 2D air-liquid interface culture

Hi all, I'm growing primary airway bronchial epithelial cells in 2D ALI culture, and plan to do cell sorting for downstream proteomics/phosphoproteomics. Does anyone have experience with this who...

01 January 1970 4,647 0 View

Bias towards a DNA/RNA understanding of drivers of biology due to superior techniques

Dear all, I've had thoughts that our understanding of what drives the biology underlying health and disease may be skewed towards DNA and RNA level alterations. Aside from the central dogma of...

01 January 1970 7,410 3 View

Human airway basal cells - subtype specific markers

Hi all, I've been using p63 as a target for immunofluorescence to universally identify basal cells in my 2D air-liquid interface cultures of human airway epithelium. I'm wondering if anyone has...

01 January 1970 3,468 0 View

Suggestions for a suitable journal on the intersection of art and science

Hi all, I've developed a method of turning raw mass spectrometry data into soundscapes, and am looking to publish a paper on this. In my mind, this work falls in the intersection between science...

01 January 1970 9,471 5 View