- İbrahim Türkel added an answer:1Efflux pump detection by PCR targeting MexAB-oprM like genes is really useful or QPCR based expression of efflux gene is necessary?
I have identified the presence of efflux pump genes in only 65% of Pseudomonas and absent in 35% of Pseudomonas strains.
Where detection of the gene by PCR targeting MexAB-oprM like genes doesn't mean that they overexpressed or not?
I prefer qPCR-based quantification of expression of efflux pump gene from mRNA.
My query is what about the 35% of strains that lack either MexAB-oprM, MexXY-oprm, MexEF-oprN, MexCD-oprJ, or none of these pumps, which means only few, or resistant strains only carry efflux pump genes or all strain will carry certain efflux gene, but only resistant strains will overexpress?
Is PCR-based Efflux pump gene detection valuable or not?
Thanks in advance.
These efflux pumps encoded choromosally. So %35 of your strains must carry this pump. But some strains have lower expression then resistance form of your strains. In my opinion, real time pcr will be effective for measure the expression levels of your strains. Or you can sequence MeX genes for %35 strains by convential pcr and jel electrophoresis and blast it.
- Alvin Uy added an answer:7What are some of the possible explanations for the non-Ohmic behaviour of an ion channel?
We are currently doing a study on the non-Ohmic behaviour of α-hemolysin ion channels excreted by the bacteria Staphylococcus Aureus. According to previous studies, these ion channels does not follow Ohm's law.
I would like to know on what possible reasons that we might need looking in to for the duration of the study that could possibly explain this behaviour.
Thank you Mr. Zylbertal, Mr. Kanjhan, Mr. Rastqar, Mr. Kahanovitch, and Mr. Ilyin for your answers. They would be of big help to our study.Following
- Linda M. Boland added an answer:3Ion channel mentors: will you please take a 2 min survey at this site on Survey Monkey: https://www.surveymonkey.com/r/FL2XFYX ?
this survey takes about 2 mins and your help is appreciated.
I will find a way to post the answers to the ion channel survey.Following
- David C. Ellinsworth added an answer:2Are we any closer to identifying the EET binding site on TRPV4?
Since Bernd Nilius predicted in his 2004 AJP Cell review that this could relate to an arachidonate recognition-like sequence (residues 402-408, for mTRPV4) I don't believe this has been clarified. Or am I wrong? Apparently mutant TRPV4 lacking the predicted ARS cannot be functionally expressed.
Thank you, I am aware of this paper and am also in collaboration with Miguel Valverde on an ongoing project. This paper does not, however, further elucidate where EETs actually bind on TRPV4, even though it very elegantly describes an important facet of converging physiological signalling mechanisms of TRPV4 activation. Since posting this question I have had personal communications and come to the conclusion that the predicted ARS is the best bet, but no data exists to verify it.Following
- Kate Goasdoue added an answer:3We did a WB using ab10096 for nAChR-alpha7. It precipitated but the darkest band didn't have the predicted band size. What do you suggest?
Blots were incubated with ab10096 overnight and for 90 min for the secondary antibody. Non-fat milk was used as blocking solution.
Sometimes proteins run differently on a gel than the predicted MW due to post-translational modifications such as glycosylation - but this shouldn't change the MW by too much. As Julia said it could be a dimer if it is double the weight. Could be also that your samples have a splice variant of that gene. Saying that, that band on the blot doesn't look too specific - if you altered your ab concentrations / block to clean up the blot you might flind that band disappears. First step is a positive control then if you want to be sure you could cut out the band from the gel and sequence itFollowing
- Eleanor Martin added an answer:3Can anyone give me some advice on coexpression in yeast?
I would like to co-express a large (250 kDa) chloride ion channel, along with one of its interacting proteins (50kDa, soluble) in yeast. We have already optimized the expression of the membrane protein in yeast, so I would like to stick with the strain we have been using as this contains a protease deletion which has proved useful in reducing proteolysis of this large protein. The problem is that this particular stain is only ura3 deficient, meaning cloning into another plasmid and using another auxotrophic marker is not an option. The strain is also lys2 deficient so could I use a -ura media with alpha aminoadipate as a nitrogen source as the two selection markers? Or I was thinking of using a bidirectional expression vector such as this pBEVY one.
Has anyone tried to do something similar and would be able to offer any advice?
Hi Rui, thanks for your response. Yes the strain is lysine deficient, which does seem to be a less common auxotrophic marker. Most of the examples of the use of this as a selective marker are based on the ability of lys2 deficient strains to utilize alpha-aminoadipate as a nitrogen source, rather than through complementation with a vector? I'm also struggling to find any vectors containing LYS2?? Am I missing something?
Hi Reza, thanks for your response. I'm afraid expression in pichia is no good for our protein.Following
- David Baez-Nieto added an answer:11Is it possible to establish the reversal potential from Ca2 + channel macroscopic current, fitting a straight line to the data after the current peak?
I/V from macroscopics currents. Just taking into account the data that preserve the linearity, avoiding the last points close to the reversal potential. It has been done before?
Thanks I'll taking it into accountFollowing
- Huanting Liu added an answer:3Why are my plasmids recombining when I transform them?
I am working with plasmids containing aion channel, with the goal of eventually using them for transfection in Hek cells. The problem I am having is preparing these plasmids at the bacterial transformation stage. I have 2 different channels (both from he HCN family), on of them (HCN2) grew perfectly on the first try in XL1 Blue cells. However I am now doing point mutations on the channel (using a Quikchange kit) and I cannot get a colony that has my intact channel. Additionally I am trying to use HCN1, another member of the same family, and it is giving me similar problems to the mutation reaction. Here is what I have tried so far:
1. I am using internal channel specific primers to screen picked colonies for the presence of my plasmid. PCR of the unmated HCN2 plasmid produce a clean band of the appropriate size. PCR of the mutation reaction prior to transformation produces a single band of the right size. but PCR's of the picked colonies for the mutants do not, they show multiple bands.
2. Used Stbl2 competent cell to hopefully prevent recombination of the plasmid,but the pct's looked the same as the XL1-Blue.
3. Tried incubation at 37 and 30 degrees, and decreasing the antibiotic concentration, but still the same problem
I have tried these things with both the HCN2 mutation reaction and the wild type HCN1 plasmid and have had no luck.
Any advice would be much appreciated! Also, if there are any extra details that would help please let me know
Your problem is not due to plasmid itself recombination, it is caused by low mutagenesis efficiency . Try to read the paper on http://www.biomedcentral.com/1472-6750/8/91 and repeat your mutagenesis follow my lab lab protocol as attached if you want.Following
- Euan Robert Brown added an answer:2Artificial bi-layer composition.
Anyone out there making planar or tip dipping lipid bi-layers? I have been told lipid formulation is 4:1 POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) to POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). Alternatively some authors use only DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). Any thoughts on what’s best for reconstituting with ion channels please?
Thank you Vitaly thats a great reply!Following
- Sai Bharadwaj Medapuram added an answer:2How does body effect increase the threshold voltage of a NMOS transistor?
When substrate's voltage is made negative relative to source's voltage the p-n junction between source and substrate is reverse biased.This means that the number of negatively charged ions in the channel have increased which in turn must increase drain current but I notice the opposite
For NMOS when we bias the substrate negative to the source the depletion region of the reverse biased pn juctions of source-body and drain-body increases expanding into the channel .So more number of negative charged ions are available while the number of electrons available have decreased.So greater threshold voltage needs to be applied to compared to the case when no bias voltage is applied to substrateFollowing
- Pablo J Sáez added an answer:10What is the difference between a gap junction and an ion channel in cells?
Are they different? Or just a specialized type of any of the two.
I guess by now you have received very detailed answers about the funcion and properties of gap junction channels (GJCs).
Just to add to the very compelling answer given by Dr Skatchov I would like to add that between the "large" molecules that permeate GJCs (Figure: http://goo.gl/4wHjEj) are small peptides (http://goo.gl/A6u1eN), microRNA (http://goo.gl/C8CGft, http://goo.gl/qwQbTI) and siRNA (http://goo.gl/Pxlya5) and not only small molecules (IP3, cAMP, etc) and ions such as Ca2+.
- Qingyao Huang added an answer:8Could someone help me to clear up the question about TTX and ICAN?
Effect of TTX on calcium-activated non-specific cation current makes me confused recently. I have no idea whether the channel activity can be blocked by TTX in direct or indirect ways.
Thank you who will give me a hand!
Thanks for your help Asaph Zylbertal.
It is reported that ICAN is responsible for the generation of pacemaker activity related to respiratory rhythm, on the grounds of cadmium and TTX sensitivity, as well as the riluzole insensitivity. However, I don't know why the likelihood of voltage-dependent calcium channels is excluded if the vdcc current is sensitive to cadmium and TTX, and insensive to riluzole too. On the other side, if the vdcc current is not sensitive to TTX, what mechanism mediates TTX to inhibit ICAN, also through inhibiting calcium influx ?
Perhaps, the location of putative contributors is concerned with the effect of TTX on ICAN as well,
I think I should search for more basic knowledge of CAN channel.
- Serguei N Skatchkov added an answer:1How do I propose an analogues for a compound like imidazopyridine?
By knowing the inhibition of carcinoma cell growth, hERG ion channel inhibition, and the structure of imidazopyridine.
indeed pyridine, pyridinium, based compounds are widely used trying to suppress cancer growth. This is attached (see please, Tobo et al., PLos ONE, 2015) article on different structures of such compounds.
When we used pyridin(ium) based compound ASP+ we visualized that this fluorescent compound is selectively taken up by astrocytes, but not neurons (except few neurons in VTA area, Inyushin et al., 2013-Fig.2). So, the compound can be very selective for astrocytes in cortex and hippocampus, or may be in other cell type in different organs that you can test. (please see attached Inyushin et al., 2010 to get some ideas).
However, most surprising was our discovery that the transporters which are taking up such compounds are relocated in cancer cells from plasma membrane to nucleus membrane, and thus, the "killer compound" can not reach the cancer cell cytoplasm (See please attached Kucheryavykh et al., 2014).
Very cordially, Serguei.Following
- Robert Lindner added an answer:7Any advice on immuno precipitation from crude/unclarified lysate?
I am stuck in this situation at the moment, trying to immunoprecipitate (IP) and Co-immunoprecipitate (CoIP) partners of an ion channel that have totally different solubility requirements.
N.B. - I have functional evidence (patch clamp recordings) that this channel is present fully assembled in my cells. Also, the two partners of this channel are part of other channel complexes (<10% form the channel I am interested in).
Protein 1: Super hydrophobic (17 transmembrane segments), I can detect it by western blot when solubilizing the PELLET in LDS sample buffer after lysate clarification. I have tried Triton-X100, CHAPS, DDM, Pierce IP lysis buffer and FivePhoton Transmembrane protein lysis buffer - none of these effectively solubilize this protein (even with additives - spermidine, glycerol). Sonicating the crude lysate helps somewhat, but this appears to disrupt the delicate interaction between protein 1 and protein 2 - i.e., I cannot see a CoIP band, but I do see a faint IP band. I can see a darker IP band when resolubilizing the pellet in lysis buffer + sonication to break up the pellet. But still no CoIP band (pellet will not re-dissolve without sonication).
Protein 2: A little more co-operative. I can detect this by western blot and IP from CLARIFIED LYSATE. I have not tried IP from the pellet for this protein. I do not see a CoIP band from clarified lysate.
My question is this: Is it necessary to clarify the lysate? I've been trying to come up with the "perfect lysis buffer" to solubilize everything, but not disrupt the interaction - but maybe I'm wasting my time? Could I just try my IP/CoIP from crude lysate? Has anyone ever tried this?
Happy to give more info on this. Thanks in advance for the help!
I also would suggest to try crosslinking on cells. Perhaps a disulfide-cleavable, water-soluble x-linker might be optimal for this purpose. This would allow you to use really dissociative lysis conditions afterwards, as long as you avoid cleaving the linker by reducing agent (include iodoacetamide or NEM in the lysis buffer). After IP, you can cleave by reduction and should be able to detect your two bands of interest. Good luck!
- Hang Li added an answer:3When the cell potassium ion channels were blocked by chemical, can they still will be detected by ion channel antibody?
I want to use 4-AP to block cell potassium ion channels, and try to confirmed with antibody to see if it is blocked or not. I was wondering even ion channel were blocked it still may can be detected by antibody, then my method won't work, anybody has any ideas? I just want to find a way to show potassium Ion channel has been blocked by 4-AP
Thanks a lot, Norbert Weiss and Sandipan Chowdhury, I appreciate all the time you spend to help me. I know electrophysiology would be the best way to do it, I just want to find another way to prove it 4-AP blocked potassium ion channel besides electrophysiology. Hi Sandipan Chowdhury, I will think about your suggestions. Thanks a lot.Following
- Alessandro Bilella added an answer:15DREADD vs. optogeneticsI am thinking of employing both these techniques in my lab and was wondering if people would like to share their thoughts/experience.
From a behavioural point of view I suppose that a major advantage of optogenetics is that you have greater temporal control over stimulation: once a dose of CNO is administered to an animal expressing DREADD there is presumably a time-to-onset and later a decline in receptor occupation and effect, whereas with ChR2/NpHR simulation is phase-locked to light stimulation.
By contrast, I would imagine that light scattering (optic fibre in brain)/failure of light to penetrate tissue sufficiently (stimulation of peripheral nerves in skin) is a drawback of optogenetic stimulation compared to oral administration of CNO, which has known efficacy at different DREADDs.
Any thoughts/comments welcomed!
Yes, in both cases is Cre-recombinase.Following
- Jitendra Singh added an answer:12Why are there current fluctuations in hERG patch clamp assay using port a patch (Nanion)?
I am doing hERG voltage clamp protocol using Port a patch but I am unable to get a steady current. The current fluctuations are so high that I can't even test a single conc. A brief about Port a Patch- its a semi automated patch clamp instrument where solutions, test compounds/ vehicle/ positive control all are added manually. This patch clamp has been recently installed in our lab and has passed the initial validation tests.
I have checked the pulse protocol and other settings but somehow I am not able to rectify this problem. Also I am using inducible CHO-TREx hERG expressing cell line. Kindly suggest..
who is the supplier of CHO-TREx hERG expressing cell line. make sure that it is expressing the herg channel by some florescence methods.
Proto col suggested by the Marry is ok u can try making following changes to your protocol
Protocol Voltage (mili-volts) Duration (mili-seconds)
Holding potential -80 50
depolarization +40 2000
depolarization +40 2000
Holding potential -80 200
Measure the current in 4th segment (+40 mV to -40 mV).
Also write in details how you are culturing the cell specifically media and antibiotic anti mycotic.Following
- Ulises M. Ricoy added an answer:19What are the simplest electrophysiology experiments one can perform in the lab, for undergaduate students?What are the simplest electrophysiology experiments one can perform in the lab, for undergaduate students?
There are several compilations references for ideas on experiments utilizing invertebrates (low cost, accessible). Here are some excellent examples for places to start:
1) Charles Drewes PhD (Iowa website) http://www.eeob.iastate.edu/faculty/DrewesC/htdocs/cddpubsweb.htm
2) Experimental neurobiology: a laboratory manual by Bruce Oakley and Rollie Schafer ; [ill. by Ken Cassady]. Published 1978 by University of Michigan Press
3) The Laboratory Cockroach - Experiments in Cockroach Anatomy, Physiology and Behavior Spiral-bound – December 31, 1981by W. J. Bell (Author)
4) Journal of Neuroscience Education, Advances in Physiology Edcuation, CBE Life Science Education, American Biology Teacher,
5) Robin L Cooper PhD (U Kentucky website)http://web.as.uky.edu/Biology/faculty/cooper/Bio450-AS300/Bio476-Spring2015.htm
6) Richard Olivo PhD (Smith College website) http://www.science.smith.edu/departments/neurosci/courses/bio330/labs.html
7) Bruce O'Gara PhD (Humboldt State website) http://users.humboldt.edu/bruceogara/Syllabi/Zoology%20310%20Lab%20Syllabus.pdfFollowing
- Babak Vazifehkhah Ghaffari added an answer:10How can I get the current equation of KCa channel in a conductance-based model?
As I know, to get a current equation for any channel, the dynamic-clamp technique is applied to the single neuron. The effect of all other channels is removed by specific blockers to get only the desired channel dynamics. KCa channel is dependent to calcium channel. So, how does the calcium channel blocked without blocking the KCa channel?
Sorry Ameera, maybe, I could not explain it well. My question was one, which Daniel Hass answered it, comprehensively.Following
- José L Lanciego added an answer:5Has anyone had any success with Duolink's proximity ligation assay (PLA) in fixed spinal cord tissue?
Hello, I'm trying to get this technique to work on K ion channel subunits in murine 4% PFA fixed spinal cord slices that have been treated with Na Citrate for 30mins at 70 degrees and blocked for an hour with donkey serum. Primary antibodies are raised in mouse and rabbit and work well in normal IHC and what we expected to work well as a positive control is not working either-has anyone got any suggestions or had issues with this kit before?
Many thanks for your reply. If you have any further question, please feel free to contact me at any time.
All the best and have a nice day, Jose L.Following
- Hannah R Malcolm added an answer:8Do ligand-gated ion channels (which can be activated from the cytosol) exist in bacteria? What is their name? In which organism?
For a project within the iGEM competition, we are willing to construct a new biological biomarker detection system. For a read-out, we are considering an electric readout. Therefore, we want to stimulate the opening of ion channels upon a secondary messenger signal. Most bacterial ion channels we have found so far are uniquely stimulated by an extracellular ligand.
We are happy for all suggestions upon this topic and the expertise in the field.
The bCNG channel family has members found in over 180 different genomes, we predict that these gate in response to cyclic nucleotides in the cytosol. These channels are a part of the MscS Superfamily of ion channels, of which some members are predicted to have different binding domains. Here is the first paper on bCNG channels.
- Keli Peng added an answer:4Can anyone advise me on the use of LaCl3 as a Ca2+ ion channel inhibitor to study Arabidopsis primary root?
Firstly, I dissolved the LaCl3 in water. And I found LaCl3 can significantly decrease the pH of 1/2MS medium when high concentration. When I adjust the pH back to 5.8, La3+ become sediment. So can anyone give me some advice?
Hi Kenji, I use KOH to adjust pH instead of phosphate buffers. Maybe it's because that I didn't add MES to steady the pH of medium. And I will also try Tris and HEPES to adjust pH. Thanks for your advice!Following
- Xin Yang added an answer:2When do we use Tonic activated NMDA receptor current?
When do we use Tonic activated NMDA receptor current?
Does this correlated to epilepsy?
tonic response related to the network synchronization or desynchronization? i just forget it. And the epilepsy also connect with the sychronization and NMDAR. Maybe there exist some relationship. And as described by Paraskevopoulos, it is too small to record it.Following
- Wei wu added an answer:7What can be the reason for the poor solubility of potassium channel expressed from Pichia pastoris?
I am trying to purfiy a human potassium channel expressed from Picha pastoris. The carboxy terminal of the protein is linked with a GFP tag for measuring the level of expression or purification . Now I have some trouble in solubilizing the protein by detergents.I break the picha pastoris with french press (1600bar, 4 times).After ultacentrifugation, I collect the crude membane, resuspend it (Resuspension Buffer: 50mM Hepes, pH7.5, 200mM NaCl, 5mM imidazole)and try several different detegents, like DDM, DM, OG,NG,LDAO(all from anatrace) to solubilize the membrane,the final concentration of detergent is 1%,and the total membrane protein concentration is about 3mg/ml. Incubate the mixture at 4 degree for 1h with mild agitation(I have tried the incubation overnight, It's just a little better).After the incubation, I centrifuge the mixiture at 160,000g for 30mins. Before/after the ultracentrifugation, I measeure the fluorescence intensity and the result is below:
the fluorescence intensity before/after ulta-centrifugation:
The effect of solubilzation is very poor, I have tried several time, the result is same.
Has somebody an idea to solve this problem? or what can be the reason for this poor solubilization?
Thank you for your help
I have solved the problem, I use the Constant system (40k psi) to disrupt the yeasts, the effect of solubilization is goodFollowing
- Ivan Lorenzo added an answer:3Is there any kind of commercial isotonic solution -with and without calcium and the same osmolarity- to carry out life calcium experiments?
Dear all, in order to perform life calcium imaging experiments I would like to use commercial isotonic solutions available in the market instead to prepare them by myself.
I believed that HANKS' balanced salt solution (HBSS) was an option. However I cannot find the same solution just with or without calcium, without affecting the Mg content. I find Ca and Mg are always coupled in HBSS.
Check LifeTechnologies as example: https://www.lifetechnologies.com/nl/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/balanced-salt-solutions/hbss-hanks-balanced-salt-solution.html
Please, any suggestion?
I will go for the HBSS without Ca+Mg. I will add the right amount of Mg. So, starting with the same solution, in case of Ca ions are needed, I will add them. To get solutions of the same osmolarity, I will add NaCl to compensate the final osmolarity. The total amount of NaCl will be negligible. Thanks both, Tamas and Manoj.Following
- Shashank Chavali added an answer:4How to calculate the diameter of an ion channel in VMD?
I ran a molecular dynamics simulation of an apo-ion channel for 15ns on NAMD. I'd like to look at the diameter of the ion channel at different intervals to examine the opening and closing of the channel.
Thank you very much. That was really helpful.Following
- Jorge Parodi added an answer:1Can anyone give me some simple cardiac cell models that incorporate the mechanosensitive ion channels?
Thank you very much.
is hard, but if you like described the ion channels characteristic, maybe you can used oocytes, for expresson, and used perfusión system for estimulationFollowing
- Taylor R Fore added an answer:3How do I perform patch-clamp recordings from dorso-longitudinal flight muscles ( in drosophila?
is there someone familiar with patch-clamp recordings from Dorso Longitudinal flight Muscles (DLM) in drosophila? I'm trying without success yet and every suggestion, papers and tricks are absolutely apreciated. Till now seems impossible to make the seal and thus record something from the fiber. I wish to thank anyone in advance for any help.
I have no experience recording from the DLM, but have you read any of Marcus Allen or Tanja Godenschwege's papers?
Also check out, if you haven't already, chapter 13 from Drosophila Neurobiology: A laboratory manual (PDF: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2946074/ ) for a quick review of this technique with references.
Best of Luck.Following
- Ivan Lorenzo added an answer:6What is the suitable calcium indicator for nuclear calcium imaging?
I want to carry out calcium signaling experiments in MSC cultured in 3D scaffolds.
Due to equipment constrains, I only can exite the fluorocrome probe above 450nm. So, I am considering to use Fluo3/FuraRed. However, I am concern about how to place the calcium indicator in the proper compartment of the nucleus. Then, I also consider to use calcium-binding fluorescent indicators like cameleons, pericams, or aequorins fused to a Nuclear Localization Signal (NLS).
Thank you all for your commentaries and suggestions.
As I would like to work with primary cells, I would go for small fluorescent probes such as Fluo4.
Why it is better the engineered and targeted protein Ca indicators than the small dyes when both can be compartmentalised and ROI selection can be done?Following
About Ion Channels
Gated, ion-selective glycoproteins that traverse membranes. The stimulus for ION CHANNEL GATING can be due to a variety of stimuli such as LIGANDS, a TRANSMEMBRANE POTENTIAL DIFFERENCE, mechanical deformation or through INTRACELLULAR SIGNALING PEPTIDES AND PROTEINS.