30 Questions 13 Answers 0 Followers
Questions related from Neal Edward Beeman
Hello everyone and I hope I find someone who has done this. Has anyone transfected THP-1 cells in suspension culture? If so, I would love a protocol.
13 August 2023 7,354 0 View
I would appreciate help here in avoiding re-inventing the wheel. Has anyone used AAV to transduce THP-1 in suspension culture? I do not want to do this in activated adherent (macrophage-like)...
13 July 2023 4,592 0 View
We have a protein of interest that seems involved in membrane blebbing. We wish to inhibit membrane blebbing in some of our experiments without inhibiting ROCK/Rho to myosin light chain kinase...
28 January 2023 3,571 0 View
Hello everyone and thanks for reading his question. I found during bacterial protein production that I had set the centrifuge much higher than intended when pelleting intact bacteria. My...
07 December 2022 6,819 1 View
Hello everybody, I am trying to cross link my protein of interest to see if it homodimerizes when phosphorylated and does not when not phosphorylated. We can stimulate autophosphorylation in...
01 December 2022 4,631 1 View
I have a protein that seems to relocate from the cell surface to an intracellular compartment when phosphorylated. When not phosphorylated the same protein appears to be associated to the cell...
16 September 2022 3,148 1 View
I am using shRNA. The construct is transfected into cells and I think that the shRNA keeps expressing from the DNA vector. I am new to this. How long following transfection should I wait to...
29 August 2022 222 2 View
I am trying to create a lipid milieu in a test tube. I need to pipette in various lipids in chloroform and then dry off the chloroform. If anyone knows the standard stock concentration of...
23 August 2022 707 0 View
Hello I am trying to find a website or freeware that predicts amphipathic helices from AA sequence. I have been trying AMPHIPASEEK (AMPHIPATHIC IN-PLANE MEMBRANE ANCHORS PREDICTION),...
08 June 2022 5,438 1 View
I am doing a fairly large panel of transfected genes of interest and also comparing Latrunculin-A actin depolymerization to cytochalasin-D actin depolymerization. I am hoping to skip some...
22 March 2022 5,088 1 View
Please see the picture below. As shown one base in my primer does not match the template sequence. Is there a rule of thumb for how many bases should match the template 5' (upstream) of the...
14 December 2021 8,017 3 View
I have used the same anti GFP antibody several times now for western blotting. I collected after primary incubation and kept at -20. The antibody was in TBS-0.1%Tween-20 and 5% non-fat dry milk....
13 December 2021 9,788 7 View
I have 2 treatment conditions. I am trying to show that in one condition my protein is randomly distributed in the cytoplasm and in the other condition is at the membrane. We are taking...
21 October 2021 8,239 1 View
I overgrew my bacteria according to OD. I just removed the seemingly correct volume and replaced with LB to get into the ideal density. I then added IPTG..Should this work?
15 September 2021 8,350 1 View
I have a bunch of Z-stacks that have a red, green, and blue. I want to merge the red and green signals only and have a maximum intensity projection of the combined output of red and green only,...
10 September 2021 6,579 1 View
Ideally I would like to be able to image live and also to be able to make fixed slides. I want to probe for the cellular distribution of the variously phosphorylated phosphatidylinositols. I...
06 July 2021 9,359 6 View
I am looking hopefully for numbers here: type of plate used, volume of lysis buffers used at each step, formulation of lysis buffers etc. I have found protocols for hypotonic lysis of...
11 May 2021 7,242 1 View
I have a small flask with ~100ml of Amp resistant bacteria in LB [Luria-Bertani] broth, that has been at 4 degrees tightly sealed for 26 days. I need to grow a larger culture of this same...
30 April 2021 1,535 5 View
I am working with a kinase that seems (by fluorescence imaging) to shuttle back and forth from membrane localization to more cytoplasmic localization. We are able to manipulate this localization...
23 April 2021 9,484 2 View
Hello and thank you all for this resource. I have routinely used fluorescently conjugated phalloidin to stain actin cytoskeletal elements for years, in fixed, permeabilized cells. Are there...
06 January 2021 6,396 4 View
I have in the past worked with the MitoTracker dies. They are rather expensive and that is the bit I am hoping to avoid. I would really appreciate it if anybody can suggest a mitochondrial...
06 November 2020 5,863 1 View
I am confident and reasonably competent with both immuno-staining and co-IP. My immunofluorescent co-staining indicates that my protein of interest does bind the actin cytoskeleton. I have...
19 August 2020 9,564 3 View
I just got back Sanger sequencing data of overlapping ~800bp regions. I am trying to stitch them together into one consensus sequence. Please point me at something free and easy that can take...
12 June 2020 709 1 View
I used to do this kind of thing and now have forgotten strategy please help and be specific. The vector is now linear having been digested with EcoRI and MfeI, then treated with CIAP, to prevent...
24 May 2020 2,576 1 View
We've certainly got the Miltenyii reagent for blocking Fc receptor. But I don't want to use it if THP-1 cells do not need this blocking. Also, any THP1 flow tips or experiences will be well...
20 November 2019 5,826 3 View
Catalog number: 4367659 Power SYBR Green PCR Master Mix. This is NOT POWER UP that I am asking about. Can I set up a plate Monday the 28th in the afternoon and run it Tuesday the 28th in...
25 May 2018 8,756 5 View
I am doing a series of co-stains in paraffin sections of various mouse tissues. I want to keep using a panel of donkey secondary antibodies that is performing very well and yes I block in...
26 August 2017 8,742 1 View
RAW 264.7 cells mouse macrophage phenotype transformed cell line. I want to transfect RAW 264.7 cells with plasmid DNA for fluorescent reporter expression There are 2 different reporters under...
03 June 2016 7,578 0 View
Can any please one post me back a protocol for generating total cell lysate from adherent macrophages (RAW 264.7 mouse cell line). I need a minimum of 10ug protein and minimum concentration of...
05 May 2016 3,979 2 View
I need to establish a cell line that will express a bicistronic reporter. I have the reporter expression plasmid in hand. I have transiently transfected this plasmid into mouse macrophage...
01 January 1970 5,509 1 View
