73 Questions 15 Answers 0 Followers
Questions related from David Schad
Hi! I want to generate basolateral-out spheroids of Caco-2 cells. I have Matrigel at my disposal. I would appreciate any suggestions or protocols. Thanks!
25 January 2025 4,804 1 View
Hello, Does anyone have any tips for lysing cells for western blot in transwell inserts? I am using 6.5mm polycarbonate inserts with 8um diameter pores (Corning 3422). I am currently lysing in...
14 January 2025 9,034 3 View
Hello, The monkey virus SV40 contaminated an unknown number of batches of the Polio vaccine administered between 1955 and 1963. Since then, SV40 has been found to transform cell lines and...
08 January 2025 7,055 4 View
Hi! I am growing intestinal epithelial cells in 24-well transwell plates with 8um pores. I am using Caco-2 or T84 cells. I find it difficult to visually assess the monolayer using light...
20 August 2024 8,783 5 View
Hello, I want to use a commercial preparation of recombinant PNGase, but I do not have the buffer. What cofactors or buffer conditions does PNGase F require? I will add the enzyme to cell...
09 February 2024 6,155 1 View
Hello, For many phosphorylation studies of MAP kinases, cells are serum-starved overnight prior to stimulation. Serum-free media such as DMEM 1X is supplemented with 0.5% bovine serum albumin....
29 January 2024 9,556 1 View
Hello, In recent years, Dawn dish soap has advertised their product by showing that it can be used to save ducklings that have been impacted by oil spills. However, detergents like Dawn work...
28 January 2024 8,007 4 View
Hi Everyone! I am currently designing primers to validate my CRISPR-KO mammalian cell line using TIDE. The primer is designed about 250bp upstream of the PAM site. Then, Sanger sequencing is...
28 January 2024 2,260 1 View
Hello, The Rac proteins are a subfamily of the Rho GTPases. The Rho GTPases are a subfamily of the Ras GTPases. Does the name Rac bear any significance regarding the protein's identity,...
21 September 2023 5,109 1 View
Hello, I am designing a plasmid with an SV40 promoter-driven antibiotic resistance. Does expression from an SV40 promoter require a TATA box upstream of the transcription start site? The...
09 May 2023 1,806 4 View
Hello, I am measuring multiple different parameters in cells using fluorescent channels in flow. I would also like to assess well viability. Given that I take the same volume from each well,...
17 April 2023 2,711 4 View
Hello, I am running a sandwich-type ELISA where I am trying to inhibit a protein-protein interaction using small molecules. For each experiment, I determine my Z' (Z-prime) to determine the...
24 February 2021 7,922 1 View
Hello, I am performing a western blot of my SARS-CoV-2 Spike-pseudotyped lentiviral particles. I denature my virus by adding concentrated virus to 2X Laemmli buffer at 1:1, then boiling for...
18 February 2021 5,292 4 View
Hello, I want to control the temperature of my ELISA plate during incubations to get consistent results. In general, what is the best temperature to coat the plate with antibody? I have been...
17 February 2021 3,128 7 View
Hello, I am western blotting SARS-CoV-2 spike protein from lentiviral using an antibody against S1. To prepare samples, I mixed concentrated lentivirus 1:1 with 2X Laemmli buffer and boiled...
08 February 2021 4,046 4 View
Hello, I have recently run a western of Vero cells for ACE2. I detected ACE2 at the correct size, but it presents as a double band. The sample was prepared by lysing cells in 1X Laemmli...
08 February 2021 7,382 3 View
Hello, In my sandwich-type ELISA which is testing for small molecule inhibition of a protein-protein interaction, it seems that DMSO percentage and dilution protocol are important. I...
01 February 2021 7,282 1 View
Hello, I am conducting a sandwich-type ELISA. It is to see if small molecules can interfere with a protein-protein interaction. As this interaction would occur in the human body, may I heat...
01 February 2021 4,672 3 View
Hello, I hope to insert a gene into mammalian cells in culture using CRISPR/Cas9 and an HDR template. Will insertion of a 1 kilobase gene cause chromosomal abnormalities and effect cell...
01 February 2021 2,888 1 View
Hello, I wish to knock-in a gene to a certain locus of a mammalian cell line using an sgRNA, Cas9, and HDR template. I will first transduce cells with the sgRNA and Cas9 in a lentiviral...
22 January 2021 6,730 3 View
Hello, Most hand sanitizers contain 60-80% alcohol. Alcohol destroys organisms including bacteria, fungi, protists, and enveloped viruses by breaking down the plasma membrane. In recent...
17 January 2021 7,240 11 View
Hello, I want to insert a gene into my cells using sgRNA, Cas9, and an HDR template. The gene that I will insert into has its own promoter. Because I will cut the gene downstream of its start...
15 January 2021 1,520 2 View
Hello, I typically harvest my lentivirus in the supernatant from packaging cells 48 hours after the media change from transfection. Last week, I forgot to harvest and ended up harvesting...
07 December 2020 2,807 4 View
Hello, SV40 is not considered an oncogenic virus in this review:Oncogenic viruses and cancer Why is SV40 not considered an oncogenic virus when it produces the large T antigen, which is...
05 December 2020 8,629 5 View
Hello, I want to target a specific gene sequence using CRISPR-Cas9 to induce the death of the cells carrying that gene. I think that there are three basic approaches: 1.) Induce a frameshift...
21 November 2020 4,815 3 View
Hello, This citation is used on Wikipedia when explaining that the identity of the parents of the fetus from whom HEK-293 cells were taken is unknown. The link to the USFDA in the references...
08 October 2020 7,976 7 View
Hello, I understand that the cell line HEK-293-FT grows faster than HEK-293-T. Is this the only difference between these cell lines? Is it correct that neither of these cell lines have...
07 October 2020 5,554 2 View
Hello, I have used an optional wash step in my miniprep said to remove endonucleases from DNA grown in strains of E. coli which produce endonucleases. The wash buffer was pre-warmed to 50C,...
07 October 2020 10,116 4 View
Hello, I am running a sandwich-type ELISA for which the readout is absorbance at 450nm. The ELISA measures protein binding which I am screening for compounds to inhibit. The 570nm...
06 October 2020 10,031 6 View
Hello, I have a lentiviral vector which I hope to package into virus by cotransfection with an envelope plasmid into a packaging cell line (293FT). My lentiviral vector is in stbl3 E. coli,...
06 October 2020 9,767 4 View
Hello, I keep my mammalian cells in a typical incubator at 37C with 5% CO2. I fill the water bath with diH20. Following the advise of a colleague, I add a few drops (250-500uL) of 20% SDS to...
03 October 2020 9,499 3 View
Hello, I am comparing the two products linked below: Pierce Cypridina Luciferase Flash Assay Kit and Pierce Cypridina Luciferase Glow Assay Kit. As I understand it, the only difference is...
18 September 2020 1,703 1 View
Hello, I am using a lentivirus encoding CLuc to transduce target cells. I want to then lyse the cells in the presence of luciferin and measure luminescence. The kit which I had hoped to use...
16 September 2020 6,973 2 View
Hello, Some of the original Polio vaccines in the late 1950's and early 1960's contained the monkey virus SV40. While these vaccines were successful in stopping the spread of Polio, many...
23 June 2020 7,854 0 View
Hello, I am using Graphpad Prism to generate a standard curve of data. In this instance, I have used the curve called "Sigmoidal, 4PL, X is concentration". Unlike for a linear regression,...
01 June 2020 2,360 0 View
Hello, I am currently performing a small molecule screen. The screen is to find a molecule which competitively inhibits the binding of two proteins. The screen is a sandwich-type ELISA. The...
21 May 2020 8,498 0 View
Hello, I am examining the phosphorylation of the MAP kinase ERK by immunofluorescence. In order to do this, I understand that serum-deprivation of cells will eliminate background...
18 May 2020 2,498 6 View
Hello, I am currently optimizing an absorbance-based ELISA assay. The ELISA is a sandwich-type, with the final component being a secondary antibody fused to HRP. I then use Turbo-TMB to obtain...
11 May 2020 4,170 6 View
Hello, Many protein shake powder mixes contain digestive enzymes such as protease, lipase, and cellulase. However, the products do not detail from which organism the proteins are produced or...
22 March 2020 5,241 3 View
Hello, I am wondering if it is alright to reuse my antibodies for immunofluorescence experiments. I have reused primary antibodies with success for the first two experiments, but then I saw a...
11 February 2020 467 3 View
Hello, I am attempting immunofluorescence of a membrane-bound, phosphorylated protein. I typically use PBS in my blocking buffer. I also use PBS and PBST to wash. I have been largely...
23 November 2019 7,992 2 View
Hello, I am performing immunofluorescence of HepG2 cells. I believe that some of my stocks may be contaminated with Mycoplasma because when I stain them, their membranes and portions of their...
23 November 2019 2,913 3 View
Hello, When utilizing bleach for biological applications, is it advisable to pour excess down the drain? What is the ecological impact of bleach entering the septic system? Is bleach...
26 February 2019 2,954 3 View
Hello, Is it possible to observe Brownian motion of particles with a light microscope at 40X? I am asking for reasons relating to mammalian cell culture. Thank you.
20 February 2019 4,571 3 View
Hello, Recently I performed lentiviral transduction on several cell lines. On day one with the lentivirus, I observed black aggregates moving (dancing, vibrating) around my cells. These are...
20 February 2019 8,601 5 View
When I freeze stocks of lentivirus, I take 2 mL of virus-containing media per cryotube and put them at -80C. How long will the virus stay viable? I have used them successfully after a month, but...
08 May 2018 3,381 3 View
In my human liver cell lines, at 10x I see many black dots floating in the media. At 20x, I see them moving around (but just within a small area). They are basically just hanging out outside of...
12 April 2018 5,848 3 View
After transducing cells with a lentivirus and passaging them with the proper selection drug, can they stop producing a transduced gene of interest? I have transduced cells to express a protein and...
11 April 2018 9,902 3 View
When I use dithiothreitol (DTT) in my western blot Laemmli buffer, I usually wear a mask. Is this entirely necessary? Sometimes I get a whiff of the DTT even while wearing a mask. Are there...
10 April 2018 6,461 8 View
When growing human hepatocytes in culture, I have always added 1% penicillin/streptomycin to my DMEM/F12 media. I recently read that this may make my cells vulnerable to mycoplasma infection. Is...
02 April 2018 5,138 7 View
For western blots, I dilute my primary antibodies between 1:1000 and 1:2500 in Licor blocking buffer diluted 1:1 with 1x PBS. How much sodium azide crystal is needed per mL to adequately preserve...
02 March 2018 552 2 View
It is necessary for me to save my PVDF membranes from western blots in order to reprobe them with different antibodies in the future. Currently, I keep my membranes in 50 mL tubes filled with 1x...
01 March 2018 4,506 11 View
When I mix one part methanol with two parts water, the bottle becomes warm. What is taking place to cause this change?
16 January 2018 649 3 View
I understand that bleach kills bacteria, archaea, and eukaryotes because it is extremely toxic to the cell. But how does bleach neutralize a virus? Or does bleach only kill the reservoirs of...
08 January 2018 949 3 View
When running a western blot, I usually load less ladder (6uL) than any of my samples (20uL). Someone recently told me that I should add Laemmli buffer to my ladder so that it is loaded at an equal...
02 January 2018 659 7 View
What is the difference between using phosphate buffered saline with tween 20 vs tris buffered saline with tween 20? I ask this for the application of performing a western blot as well as...
24 December 2017 9,188 2 View
Most protocols suggest using a lysis buffer, spinning, and then adding Laemmli buffer to the supernatant. However, I have heard that it is easier to simply lyse the cells with 1x Laemmli buffer,...
21 December 2017 1,333 4 View
On Thermo's site it says that cationic lipids mediate entry into a cell via endocytosis. Why does the cell uptake the lipid-plasma complex? After entry, how does the lipid-plasma complex escape...
18 December 2017 5,972 1 View
When picking single clones, I use cloning cylinders. A problem which I have faced is that when I use 100 uL of trypsin per cylinder, no cells adhere when directly transferred to a 48-well plate. I...
11 December 2017 9,235 2 View
When I wash cells with PBS, what happens? Why does PBS sometimes cause confluent cells to peel off? Is washing with PBS always necessary before splitting or freezing cells? What is the functional...
08 December 2017 4,437 3 View
Suppose that a bicistronic mRNA is being translated and that the first gene is preceded by a 5' cap while the second is preceded by an internal ribosome entry site (IRES). If a ribosome begins...
08 December 2017 9,943 1 View
I believe that some forms of radiation can denature proteins. But can any sort of electromagnetic radiation actually cause changes in nucleic acid composition? Specifically, can portions of bases...
07 December 2017 6,984 5 View
In the NIH format of writing up an experiment, the aim, methods, results, etc. are clear and follow one another in order. This standard of writing is good, but it is not always feasible in the lab...
07 December 2017 8,241 5 View
What is you favorite gel box? And what is your favorite transfer box?
07 December 2017 7,029 6 View
When one sterilizes media, how do its protein constituents remain intact? Microorganisms that were in the media are presumably destroyed by protein denaturation. Are full proteins present in the...
07 December 2017 5,508 6 View
Does bleach simply denature or nullify any protein that it comes into contact with? Is there a recommended period of time to let bleach sit on contaminated flasks and tubes? Is it true that...
07 December 2017 9,935 4 View
I have heard that between 24 and 48 hours is a good length of time. I plan to add the virus to fresh media with serum and antibiotics, wait 48 hours, and then change out the media. I am performing...
06 December 2017 928 1 View
At what point is an object so small that it cannot reflect photons and therefore have no color? Are large proteins large enough to have color? What about small prokaryotes?
06 December 2017 4,971 4 View
What prevents a single-round lentivirus from transducing the cell line in which it was generated? I am using GP2-293 cells as a packaging cell line to create lentivirus with the VSV-G surface...
01 December 2017 10,041 4 View
I ran the gel at 60 volts, and then 100 volts. By the time of completion the entire box was warm . I had it filled in and around the dam with about a liter of running buffer.
01 December 2017 5,391 3 View
As far as I know, three doses of the HBV vaccine are administered after birth. Why is a booster not required years later to maintain immunity to HBV? I ask this because HBV is said to have genomic...
01 December 2017 2,278 4 View
Some of my packaging cell line (GP2-293) have peeled of into the media containing the lentivirus which they have produced. If I spin down the tube, will the lentivirus also go to the pellet? I...
30 November 2017 4,709 0 View
What is the proper way to dispose of a 20% methanol solution?
28 November 2017 163 6 View
