There are two different cell lines but we do not know that these cell lines have Gs or Gi proteins, associated with their G-protein coupled receptors. If we wants to know about this. Can we design a experiment through which we would be able to identify specific Gs or Gi proteins in cell lines?

  • $\begingroup$ You can design such experiments. Do you know which receptor you're interested in? What have you learned from the literature about how GPCR signalling pathways are evaluated? $\endgroup$ – user560 May 9 '14 at 22:17
  • $\begingroup$ I have studied about Adrenergic Receptors. Beta Adrenergic receptrs and alpha2 Adrenergic receptors.. ? $\endgroup$ – katherinebridges May 10 '14 at 3:32
  • $\begingroup$ Wat else do you know about your cell line? Are you sure that the two receptors are expressed in the cells in a mutual exclusive way? $\endgroup$ – Chris May 11 '14 at 14:44
  • $\begingroup$ I want to know that how can i distinguish between these two cell lines that whether they are Gs or Gi cell lines associated with the G protein coupled receptors..? Beta-Adrenergic receptor (epinephrine) is for Gs and alpha-adrenergenic receptor (acetylcholine) is for Gi.I have this information about the two cell lines.. $\endgroup$ – katherinebridges May 11 '14 at 14:49
  • $\begingroup$ Thats why I ask if you know if these proteins are expression mutual exclusive. If they are, you can do a simple RNA extraction, cDNA preparation and then do PCR (doesn't have to be realtime PCR) with specific primers to sort this problem. $\endgroup$ – Chris May 11 '14 at 20:20

Bez experiments are too difficult and costly you don't need to do that.

Take, label samples then apply ligands and sustenance. The inhibitory will have have a slower rate of growth not death and use a control with replicates. Also if you have the money buy an epac FRET to measure cAMP. Find someone to do the FRET if you can't. It will take a longer than a day but you avoid the whole I gave it too much it was going to die anyways. If you did not initially separate your cell lines by proteins and have a mix of Gs Gi within the cell lines and you need to find which ligand causes a dominant response then you will have to do individuals of these test. There are companies that specialize in growth response.

If not FRET. Find some business with an ION torrent and sequence the genome its really really cheap. If your proteins have known homology to Gs or Gi this will give the best results quickest. If you're proteins function is novel add ligands and the Gi will have less PCR amp than the Gs. Don't do your own NA extraction just let them. I've seen so many samples go bad in delivery because people tried to save a buck.

Also since you had trouble designing this experiment find a company which will run the whole of the experiment for you. Most places with ION torrents have the whole lab setup to run such an experiment and do so on a weekly basis.

  • $\begingroup$ ION torrent processing is a great option too! But regarding epac FRET, as far as I know speaking to a few friends, FRET based experiments takes a while to optimise/make them work but could be wrong as I have never done them personally. Getting a company to do this for you is always the most convenient, i agree, if it isn't part of your project. $\endgroup$ – Behzad Rowshanravan May 14 '14 at 9:39
  • $\begingroup$ @Bez yeah pay someone who has one was the theme of my answer $\endgroup$ – user1357 May 14 '14 at 9:44
  • $\begingroup$ @caseyr547 I don't understand how sequencing genomes would get you anywhere: assuming these cell lines are from the same organism then they will both have the same genome carrying genes encoding all G proteins. $\endgroup$ – Alan Boyd May 15 '14 at 14:31


Based on comments and responses my original answer to this question received I have decided to edit the answer and respond to some questions.

Since I work on proteins, I tend to favour protein based approaches specially when wondering about the presence of a protein. This is simply the case because protein is what is important functionally (purely talking about protein based problems/questions here and not talking about RNAi stuff here) so what I have experienced editors require for majority of papers, they want to see some sort of protein work. Now I'm aware that is not always possible since the antibody (Ab) might not be very specific or sensitive specially for low expressing proteins. If a protein is highly transient that makes the problem even worse, which is why many people resort to mRNA expression to make deductive reasoning for the presence of proteins although that makes a number of assumptions about how the mRNA is processed and whether the relationship between mRNA expression and protein expression level is linear but thats another discussion. So all I'm trying to say is that it's important check for the presence of a protein using Ab based techniques, which are immunohistochemistry (IHC) and western-blot (WB).

So my primary recommendation is to use Gi and Gs species specific (non-cross reactive) antibodies to check for the presence of your proteins of interest and always use Ab heavy chain control for IHC and a negative control for your WB using a sample that you know doesn't express your proteins of interest, if thats available. IHC also tells you some spatial information and whether Gs and Gi are both present in the same cell line or not and if they co-localise etc etc! Obviously check for Ab availability and if its compatible for IHC or WB procedures.

The other method that is feasible is if you know your two different cells express only one of the Gs or Gi exclusively, you can look at their specific inhibitors and add it to some of your cells and the chances are if the dose is high enough it will cause the cells to die since Gs or Gi are crucial for cell function. You can probably get away with an experiment like this using wells in a 24 well dish so you don't have to use that much media or drugs. But with stuff like this it might not work and you might get off-target effects so just do this as a complementary test. For your drug test always do a DMSO control since the chances are that's what the drugs are dissolved in and if not just use whatever solvent the drugs are dissolved in and add them to your cells in your control experiments.

Now the other possible and commonly used method to check for the gene expression of protein and subsequently infer its presence as a protein is to use a reverse transcription based PCR, such as RT-PCR although it doesn't have to be real-time as correctly mentioned in the comments. Please note that I'm not at all an expert in this and do check my answer in this section since I haven't personally done reverse transcription based PCR. As far as I'm aware, the basic procedure is to extract cellular mRNA, which can be performed using kits such as RNeasy or a similar product (I'm not promoting any products here and these are just what I have came across!). The protocols come with the kits and available online, and tells you exactly what min/max cell number/RNA levels should be present and how your cells should be (pelleted) before processing. Another method for RNA extraction is CsCl but thats pretty messy and complicated and requires high expertise to get it right but I know its inevitable for certain situations but for cell processing, a kit will do. At least RNeasy protocol states it enriches for mRNA and selectively exclude other types of RNA. Once you have your cellular mRNA you can proceed to reverse transcription using the appropriately designed primers for your gene of interest using a thermocycler. Again I'm not sure what tools to use for designing the primers for making cDNA from mRNA and what to look for (apart from the fact that make sure your primers are specific) as I only do regular PCR but would welcome any edits! Once you get from mRNA to cDNA, you can then run them on your (Ethidium Bromide or whatever DNA staining based) agarose gel and see if your amplicon is present and is the correct size (again based on the primers you have chosen you would know what the correct size is) and hence whether your gene is expressed or not. At least RNeasy kit says you do not need to use DNase digestion to get rid of any DNA before reverse transcribing your mRNA as most of it gets cleaned up when using the kit but you have that option. Now regarding the next-generation RNA sequencing I do not know anything about them so would again welcome any edits but seems unnecessary since you only want to know if the gene of interest is expressed so don't need any sequence info but thats an option too (although I imagine a little expensive?). Just to mention something very obvious, it's important that the species, which your lines have been generated from are sequenced or you have good sequence information about the Gs and Gi genes in your lines otherwise designing primers would be guess work. Its always best to see if anyone has done what you are trying to do and ask for their primers as most people are happy to share reagents.

Now you have probably noticed so far that my methods are quite biased towards molecular biology approaches and that's simply because that's what I know more about, however a great suggestion was to use cAMP levels as an indicator for Gs or Gi expression. Again I'm not an expert in this since I haven't ever analysed small molecules but I imagine that involves something like a HPLC, which you need to discuss with a chemist/biochemist how to extract and clean up your cells from everything and leave only the cAMP. There is probably a procedure/kit/biosensor for it but I will avoid getting into it as I know virtually nothing about this. However I have a note of caution for these types of indirect experiments. I have no idea what your cell lines are and how well they are characterised but since lines have all sorts of mutations and deletions and insertions in them, it's not a good idea to make inferences from cAMP levels. Since considering cAMP presence is quite important for cellular function, their absolute levels in a cell line is not necessarily a good indicator of whether one cAMP production regulator is present or not and I'm sure if you do something like this you open yourself to major criticism by any journal editor/referee. What will be a good idea though in any future experiments you do with these cells is to over express Gs or Gi and see their effects on cAMP production but I just don't recommend using cAMP levels as a sole indicator as to whether Gs or Gi is expressed or not.

If everything else fails and you got tired and wanted to wash your hands off this identification task, you can always lyse your cells and send them for mass spectrometry (MS) if there is such a facility at your institute. It's crucial that the species, which your lines originate from are characterised in terms of their global protein sequences since programs such as Mascot query the raw spectral data against databases such as UniProt to get protein coverage and subsequently protein hits. Also before you go ahead with you MS experiments, just speak to the facility manager and ask for guidance as to what lysis buffer is best to lyse your cells in and what components should not be in your lysis buffer and how sensitive their machines are and how likely you are to get detection and what your protein concentration should be. I think the best machine to use is Orbitrap and as a general rule, the higher the protein amount, the more likely you are to detect it. Since cell lysate is pretty messy and the MS machine columns can get saturated by abundant proteins such as actin, it would be best to run your lysates on an SDS-PAGE gel under reducing conditions (you can even do multiple loads by running your samples in the gel, switching off the WB machine, loading more samples and running them in the gel and continue doing this a few times. It works, I have done it myself but for pulled down samples which are a bit cleaner (less contaminants/unwanted proteins) in general). Once your gel runs through just cut the region of the gel corresponding to the molecular weights of Gs and Gi and send that for MS identification. This method (running your lysate through SDS-PAGE gel) gets rid of unwanted proteins that can interfere with the identification process. But MS is very expensive!

Again I would highly welcome any edits to my own edited reply! I'm here to help and learn.

  • $\begingroup$ As in G protein coupled receptors, the Gs protein is associated with adenylyl cyclase and thus increase the level of cAMP in the cell by activating adenylyl cyclase but when Gi protein is associated with adenylyl cyclase it will inhibit the level of cAmp in the cell by inhibiting adenylyl cyclase, so is their any technique by which we are capable to detect the elevated presence or absence of cAMP in the cells. $\endgroup$ – katherinebridges May 12 '14 at 5:30
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    $\begingroup$ based on your comment, you should edit your answer to describe making cDNA from mRNA and sequencing those, or using next-gen RNA sequencing protocols. You are correct, 'genomic' sequencing won't address expression. $\endgroup$ – PlaysDice May 12 '14 at 12:00
  • $\begingroup$ Ok, i have now extensively edited my original answer. hope the information are helpful. $\endgroup$ – Behzad Rowshanravan May 12 '14 at 17:19
  • $\begingroup$ @ Shigeta, thanks for the spelling corrections. My keyboard isn't working well for some reason. Just thought I point out it's not RNAeasy its RNeasy! But thanks to all anyways! $\endgroup$ – Behzad Rowshanravan May 12 '14 at 17:57

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