Neal Edward Beeman

30 Questions 13 Answers 0 Followers

Questions related from Neal Edward Beeman

Can anyone share a protocol for transiently transfecting non-activated or non-adherent THP-1 cells?

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 7,275 0 View

Does anyone know if Adeno-associated virus (AAV) effectively transduces non-activated, non adherent THP-1 cells?

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 4,518 0 View

Does anyone know of an inhibitor of membrane blebbing that does not work through myosin inhibition?

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 3,492 0 View

Does anyone know if centrifuging bacteria at too high of a force during protein production can cause cleavage of target protein?

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 6,757 1 View

Does anyone have a protocol for crosslinking proteins using DSS in intact, adherent HEK cells, or any adherent cell line?

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 4,567 1 View

How can I demonstrate that my protein of interest is binding intracellular plasma membrane and then relocating to the membrane at the cell surface?

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 3,107 1 View

What is a typical time course to observe the effects of shRNA knockdown of target protein?

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 179 2 View

Does anyone know the proper stock concentration for cholesterol in chloroform?

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 661 0 View

What is the best way to search from AA sequence for amphipathic helices?

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 5,384 1 View

Does anyone have a protocol for treating HEK or HEK293 cells with Latrunculin-A to cause actin depolymerization?

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 5,038 1 View

How many base pairs need to be matched 5' of the mismatched base when using a primer to introduce a point mutation?

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 7,983 3 View

How many times can I use the same antibody in a Western blot?

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 9,750 7 View

What statistical analysis should I use to determine significance in the trends shown in the attached graph?

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 8,194 1 View

Is it okay to dilute a bacterial culture with LB to lower the concentration into the ideal range during IPTG induction?

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 8,295 1 View

How do I merge only the red and green in a Z-stack with red, green, and blue in FIJI?

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 6,545 1 View

I am looking for variously fluorescently labeled probes for cellular phosphatidylinositol forms. Does anyone know a good resource or company/brand?

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 9,313 6 View

Does anyone have a simple and cheap protocol for isolating the membrane fraction Vs the cytosolic fraction of adherent cultures of mammalian cells?

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 7,201 1 View

Can I use a liquid bacterial culture that has been at 4 degrees sealed for 26 days?

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 1,471 5 View

Does anybody have a simple protocol for getting a membrane fraction and a cytosolic fraction from adherent cells for comparison by western blotting?

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 9,425 2 View

Does anyone know of probes for tubulin based cytoskeletal elements to be used in fixed cells similarly to fluorescent phalloidin probes for actin?

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 6,361 4 View

Does anyone know of a more affordable alternative to Mito-Tracker, that is well retained in fixed cells?

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 5,803 1 View

Is there a technique (aside from immuno-staining) to show that a protein of interest binds filamentous actin? I have not been able to co-IP?

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 9,508 3 View

What is an easy to use free tool for multiple sequence alignment of sequences with overlapping ends that has an easy intuitive interface?

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 654 1 View

What is a good ligation strategy with compatible ends EcoRI and MfeI in the vector DNA and the insert?

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 2,520 1 View

Do I have to block Fc receptor when antibody staining the monocyte derivedTHp1 cell line? Does anyone have a THP1 flow cytometry staining protocol?

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 5,780 3 View

Can I set up a plate for qPCR using Power SYBR Green PCR Master Mix #4367659 and run it a day later?

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 8,703 5 View

Is there an antibody (preferably not raised in donkey) that stains mouse MMR/CD206/Macrophage mannose receptor 1 in paraffin sections of mouse tissue?

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 8,701 1 View

Does anyone have a tried and true protocol for transfecting RAW cells with plasmid DNA for protein expression?

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 7,530 0 View

Does anyone have a complete protocol for generating total cell lysate for proteomic analysis from adherent tissue cultured cells?

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 3,914 2 View

Is there a protocol to establish a stably transfected macrophage cell line, preferably in mouse?

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 5,463 1 View