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Questions related from David Schad
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...
02 February 2020 6,248 3 View
Hello, Recently I asked my colleague whether we may simply microwave culture media for mammalian cell culture in order to warm it prior to use. He told me that we may not microwave cell...
12 December 2019 7,549 2 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...
11 November 2019 1,200 1 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...
11 November 2019 768 3 View
Hello, I am attempting to detect a phosphorylated protein using immunofluorescence. Should I include a phosphatase inhibitor in my blocking buffer? Will a protein's phosphorylation status...
10 October 2019 7,908 1 View
Hypothetically, could someone survive by consuming DMEM with L-glutamine? I understand this is not recommended. I just wonder, because DMEM is formulated for human cells. Thank you.
02 February 2019 8,986 4 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...
02 February 2019 346 4 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.
02 February 2019 1,293 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...
02 February 2019 3,581 4 View
In a pinch, can 70% methanol replace 70% ethanol for sanitizing surfaces prior to cell culture?
01 January 2019 3,325 13 View
If I always culture my mammalian cells in media with penicillin and streptomycin, might I be selecting for an antibiotic resistant strain of bacteria over many passages? How would I know?
09 September 2018 4,764 4 View
Hi, Do DNA binding dyes like CellTox Green (Promega) interact with DNA in apoptotic bodies? I am not sure, because in apoptosis, chromatin is condensed into a discrete apoptotic body. Also, if...
09 September 2018 5,087 4 View
Sometimes I aliquot liquid into 2 mL microcentrifuge tubes to store at -20C. The aliquot may be 50 or 100 uL. I will check hours or days later and some have not frozen. It is an antibiotic...
07 July 2018 1,862 1 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...
04 April 2018 6,684 9 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...
04 April 2018 6,149 4 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...
04 April 2018 2,433 8 View
Can I reuse 1x tris-glycine running buffer after using it to run a western? If so, how many times can I reuse it? Thanks.
03 March 2018 4,859 1 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...
03 March 2018 6,010 2 View
What do mammalian cells look like when infected with mycoplasma? Is a granulated phenotype with extracellular granules typical?
03 March 2018 6,908 3 View
Does DMSO need to be kept in a desiccator? I am worried about possible bacteria and mycoplasma contamination sources of mammalian cell lines.
03 March 2018 2,911 6 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...
01 January 2018 400 8 View
When performing a western blot transfer, why does cold transfer buffer optimize the process? Also, why is methanol added to transfer buffer?
01 January 2018 5,334 4 View
If tween 20 is a detergent that washes of antibodies, why is it sometimes used in blocking buffer. Does its presence prevent nonspecific binding exclusively? Would it not reduce the amount of...
01 January 2018 9,270 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...
01 January 2018 7,619 4 View
Technically all organic mater is composed largely of proteins. Even lettuce is full of proteins. Nevertheless, many food products are labeled as containing little to no protein. For instance,...
01 January 2018 7,024 6 View
What is you favorite gel box? And what is your favorite transfer box?
12 December 2017 8,068 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...
12 December 2017 5,454 4 View
If I have run and developed a western blot PVDF membrane, but wish to redevelop it with different antibodies, how do I strip the membrane of primary and secondary antibodies? Is it completely...
12 December 2017 6,545 6 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,...
12 December 2017 8,386 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.
12 December 2017 8,383 4 View
What component of bacteria is impacted by UV light? Is this due to DNA, protein, or lipid degradation?
12 December 2017 6,007 9 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...
12 December 2017 4,200 5 View
After I have trypsinized mammalian cells and removed them from a flask, can I reuse that flask to seed the same culture? This is assuming that I used good aseptic technique. I mainly want to do...
12 December 2017 3,583 8 View
Taking into account the estimated age of the Earth, the estimated number of snowfalls which occurred over its history, and the estimated number of snowflakes which formed in each snowfall, what is...
12 December 2017 9,297 4 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...
12 December 2017 6,397 2 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...
12 December 2017 2,262 5 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?
12 December 2017 2,471 4 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...
12 December 2017 6,618 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...
12 December 2017 2,573 3 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...
12 December 2017 3,113 1 View
Can I reuse western blot transfer buffer, or is it only good for one run? I use Novex tris-glycine transfer buffer with methanol. If I can reuse it, how do I go about filtering it between uses?
11 November 2017 6,271 4 View
Is it to denature the proteins? I know that DTT reduces disulfide bonds. Please explain to me why this step is so essential.
11 November 2017 1,408 3 View
Some protocols suggest heating, others cooling. Should the lysis buffer be ice cold? Is scraping the bottom of the plate necessary? One protocol suggests using Laemmli buffer in place of lysis...
11 November 2017 6,272 3 View
Specifically: When using three plasmids in a transfection of GP2-293 cells, in which one confers GFP, one the envelope protein, and one the gene of interest (GOI). How much P3000, Lipofectamine...
11 November 2017 9,416 1 View
Specifically: What is the optimum concentration of rat tail collagen I? How long should the collagen solution be left on the plate? Should I wash the plate with PBS? How long should drying take...
11 November 2017 395 0 View
