44 Questions 22 Answers 0 Followers
Questions related from Charles Fisher
Just a generic question, what is the average half-life of a typical membrane protein? If you have a review off hand that could be sent to me, that would be greatly appreciated.
04 April 2019 330 3 View
I have an Orion Versa Star Pro Multi-Meter and I need to get data off of it into Microsoft Excel. We are currently using Hyper Access as a logging software but this is not meeting our needs. We...
01 January 2019 6,197 0 View
I am looking for an Olympus Cx43 model microscope that has been used or was a demo model. I would also settle for other earlier models of Olympus microscopes. I need a 100X objective, a camera...
11 November 2018 4,151 8 View
Can food grade CO2 be used in place of medical grade CO2 for a cell culture incubator? Our lab is running out of medical grade CO2 and all we have on hand is food grade. We can't order more...
06 June 2018 392 0 View
Hi, I need a good inhibitor for CDC42. I am new to working with this protein and wonder if anyone knows what inhibits it well. Thank you! Chuck
04 April 2018 3,931 10 View
I am looking for a dominant negative Wiskott Aldrich Syndrome Protein Mutant. I would prefer if it has an mCherry tag. Our lab would pay for shipping and acknowledge your contributions to any...
03 March 2018 6,389 0 View
I am trying to delete 30nt regions of a plasmid but have been unsuccessful in my PCR using overlap extension PCR. My primers range in size between 23 and 36 bp in length. I fear that my deletion...
12 December 2017 4,862 6 View
I am trying to purify DNA from a gel but yields are seemingly too low. I have loaded ~500ng of DNA onto my gel, run it, and cut it out and purified it using a Quigen Gel Extraction kit. I am only...
12 December 2017 6,525 13 View
Hello, I am relatively inexperienced with certain molecular biology techniques. I have a gene that has been cloned into a plasmid for me. I would like to leave the N and C Terminal domains intact...
11 November 2016 1,148 2 View
Hello, I am attempting to use Quikchange Mutagenesis to introduce a single nucleotide mutation into a circular plasmid. I have run the mutagenesis protocol in a thermal cycler with the following...
06 June 2016 2,685 7 View
I want to get a membrane IMPERMEABLE dye into a large number of HeLa Cells in culture. What are some techniques to introduce a membrane impermeable dye into cells?
02 February 2016 2,671 3 View
I am trying to isolate primary neurons for cell culture and am wondering if there is a difference between using Penn/Strep vs. Gentamicin for my neuronal media?
12 December 2015 4,032 4 View
I want to culture rat neuronal cells but have not done it before. I am planning on using neurobasalA media supplemented with B27 and l-glutamine. After isolating the neurons with papain and...
12 December 2015 2,201 9 View
I am trying to culture primary neuronal cells and am a bit confused by the many different media formulations. Does anyone have experience culturing neuronal cells and if so, what media do you use...
11 November 2015 4,464 11 View
I am attempting to knockdown a protein in HeLa cells and observe its effect on another protein that I am transfecting into the cells. I am attempting to knockdown (KD) a protein (clathrin) in HeLa...
10 October 2015 3,024 7 View
If I would like to knock down one particular gene in HeLa cells, would it be better to use CRISPR or RNAi. I don't want to create a stable cell line, just knock down one gene. What are the...
01 January 2015 6,021 8 View
What is the size limit for a structure to be internalized into a cell through clathrin mediated endocytosis before another process, e.g. phagocytosis takes over for endocytosis? We have a large...
12 December 2014 317 3 View
I have a membrane protein with a GFP tag on its C-terminus. I have been trying to perform a co-ip on this protein by pulling on the GFP tag, but it has come to my attention that the GFP tag may be...
10 October 2014 5,603 4 View
I am expressing a protein with a GFP tag on its c-terminus. I have been unable to co-ip or ELISA with this protein using an anti-GFP antibody. I seem to get a very bright band on my gels...
10 October 2014 3,225 9 View
Is there a difference between 1% NP-40 versus 1% Nonidet-P40 for RIPA buffer for Co-IP? These are two different detergents but it is a common misconception that they are the same thing. Would one...
09 September 2014 7,275 8 View
Is there a way I can lyse cells with a GFP-construct in a mild RIPA buffer, put the lysate into a 96 well plate, and quantify the GFP fluorescence? I want to quantify the relative GFP expression...
09 September 2014 4,186 3 View
Has anyone used the Miltenyi Biotec GFP/HRP antibody (130-091-833) for ELISA? I am attempting to use it as a detection antibody for ELISA and it is not working with my GFP construct. I have tested...
09 September 2014 5,183 1 View
I have been working for months on trying to get a Co-Immunoprecipitation to work between proteins X and Y and have been unable to do so thus far. I am thinking of switching to ELISA but have no...
08 August 2014 8,115 5 View
I am doing an IP and got a peculiar result. I pulled protein X, ran it on a gel, and probed for protein X. I used the same antibody for the IP and the western. I got my protein of interest at the...
07 July 2014 5,559 4 View
I am trying to look for a physical interaction between proteins "X" and "Y" using Co-IP. X is GFP tagged that I express in MDCK cells which normally don't express it. After transfecting the MDCKs,...
06 June 2014 1,602 4 View
I have been trying for months to determine if proteins "X" and "Y" co-immunoprecipitate. However, whenever I run a negative control experiment that contains cell lysate and protein A magnetic...
06 June 2014 2,751 7 View
I am attempting to replicate a Co-IP someone else did. They had a defined positive and negative control. My positive control pulls down my protein of interest, but my negative control does as...
04 April 2014 8,436 3 View
As a lab we have been trying to look for interactions between clathrin and other proteins using Hela Cells and Co-IP. Unfortunately, it seems that our protein A and protein G beads seems to...
02 February 2014 4,401 1 View
I am trying to perform a co-ip using the µMACS™ GFP Isolation Kit but am so far having no luck. I am pulling down protein A that has a GFP-tag but when I probe for protein B I cannot detect it. I...
02 February 2014 1,687 1 View
I am trying to replicate a colocalization experiment but I am having no luck. The only difference between the previous experiment and my own is that we are using different transfection reagents:...
11 November 2013 9,139 4 View
For cell fixation, is there a big difference between using 2% or 3.7% formaldehyde or paraformaldehyde? Does such a small percentage make a big difference for immunofluorescence?
11 November 2013 9,640 30 View
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11 November 2013 1,404 36 View
I am trying to find a very specific co-localization of clathrin on hela cell plasma membranes using the X22 mouse anti-clathrin antibody, but have so far been unable to do so. I have tried using...
11 November 2013 7,773 2 View
I want to use a particular antibody for IF. Some protocols say to dissolve the antibody in 10% FBS while other protocols say to dissolve it in 2% BSA. Can the BSA or FBS make a difference for the...
10 October 2013 2,834 8 View
I am looking for the sequence to a plasmid called pBeh pac18. It was created in 1988 in the Hauser lab. It is not a commonly used plasmid and I cannot find its sequence online. Can anyone direct...
09 September 2013 3,097 0 View
Connexin/gap junction free cell line?
09 September 2013 7,459 2 View
What is the consensus motif for Disabled-2 (Dab2) binding? I am familiar with the NPXY motif but I am wondering if there are any others.
08 August 2013 8,230 0 View
I am looking for drugs or treatments that inhibit the blebbing process during apoptosis. Any suggestions would be helpful.
08 August 2013 5,290 6 View
Does anyone know of any cell lines that do not express clathrin? It would probably have to have been a line that was artificially created.
07 July 2013 6,051 1 View
I would like to attempt to isolate hepatocytes from fresh rat livers using trypsin with EDTA. I am wondering if this would be effective or if I should use collagenase.
07 July 2013 3,716 17 View
Is there any special media I should use for cells that I isolate directly from rat livers? Could I use DMEM or F-12 or do I need something specific to keep these cells alive?
07 July 2013 7,372 16 View
I am looking for a cell line that expresses Cx32. I would prefer a line that only expresses Cx32, but I would also be interested in cell lines that express Cx43 or Cx26 in addition to Cx32. Any...
06 June 2013 1,958 1 View
I am attempting to cast either 10% or 12% gels for a western blot, but every time I attempt to do so, the gel shrinks at least a centimeter from where I originally filled it to to the point where...
03 March 2013 2,166 43 View
I am curious if anyone knows of a cell line that endogenously expresses Cx26 or Cx32. I would prefer a line that only expresses these connexins and does not express Cx43.
03 March 2013 779 0 View
