- Closed account added an answer:How can I measure metabolic fluxes in cells without C13 labelled carbon substrate?
I am very new to this flux analysis technique. Does anyone know how I can calculate cell metabolic fluxes without using the labelled carbon source. I know it's quite an old technique to use unlabeled carbon, everyone is using labelled carbon for flux analysis. But due to certain limitations I can not go for labelled carbon sources.
yes my student ,i am proud of you to mention this question ,you can use the following results c fluxes, especially in complex mammalian systems that require multiple substrates. Here we have experimentally determined metabolic fluxes in a tumor cell line, successfully recapitulating the hallmarks of cancer cell metabolism. Using these data, we computationally evaluated specifically labeled 13C glucose and glutamine tracers for their ability to precisely and accurately estimate fluxes in central carbon metabolism. These methods enabled us to to identify the optimal tracer for analyzing individual fluxes, specific pathways, and central carbon metabolism as a whole. [1,2-13C2]glucose provided the most precise estimates for glycolysis, the pentose phosphate pathway, and the overall network. Tracers such as [2-13C]glucose and [3-13C]glucose also outperformed the more commonly used [1-13C]glucose. [U-13C5]glutamine emerged as the preferred isotopic tracer for analysis of the tricarboxylic acid (TCA) cycle. These results provide valuable, quantitative information on theFollowing
- Vikram Narayan added an answer:How does one do Metabolic Flux Analysis on proteomics data?
I have a large-ish dataset of the relative expression of about 8,000 proteins - I want to see what metabolic pathways change in this dataset, and how. I've been advised MFA is the way to go...can anyone point me in the right direction i.e. analysing SILAC data using MFA? Thanks!
Cheers for that guys - in response to your questions: I have SILAC data from 3 time points, from 3 biological replicates. The 8,000 proteins are across all 3 replicates in all time points and I've done extensive statistics mainly using R to narrow down my list. I've also done all the standard analyses - ORA (DAVID, ReviGo etc etc), GSEA...
Tanveer - ta for the paper; that's exactly what I was looking for! I was hoping to measure a deviation of sorts from a theoretical metabolic steady-state using my peptide/protein quantification - the paper seems perfect! I'll look through it just now.
Thanks again guys!Following
- Zhang Hao added an answer:Can anyone suggest ways of improving 13C labelling of TCA intermediates?
I am interested in studying the labelling pattern of a cyanobacteria using 13C. I do a steady state labelling with uniformly labelled 13C-NaHCO3 and draw out samples at different times points (20sec-30mins) and then detecting the metabolites by LC-MS/GC-MS. I am consisting finding that the labelling is very poor in TCA cycle intermediates like succinate, fumarate, malate, citrate etc. I get good intensities for the Mo (unlabelled) for all these organic acids (except fumarate, where I do not get any peak)> But, the M+n (labelled) peaks are of very low intensities. Can someone suggest ways of improving the labelling for these organic acids. I am able to detect labelling for other intermediate metabolites like PEP, 3-PGA etc.
may i ask you:which column did you choose for your LC-MS analysis.Following
- Marek Wojciech Gutowski added an answer:How do magnetic flux densities add-up when placed in an attracting position with an iron/steel yoke?
I am constructing a Hall measurement set-up for my research. For the required magnetic field I am using two permanent rectangular magnets facing each other in attracting positions with an iron yoke. I found that, the magnetic fields of the two magnets are going to add-up in the exact middle of the gap according to the Magnetic superposition principle. But I am not sure if the iron yoke is going to make any contribution to the perpendicular Magnetic field on the sample.
I managed to find these (http://www.magneticsolutions.com.au/magnet-formula.html#dr) sets of formulas over internet as well, but I am not very confident to rely on it. Therefore writing here, thinking if it possible to have any assistance. Thanks.
Think about a bar magnet. The strongest field it creates is, of course, near its poles. On the other hand, the B (or H) lines are always closed, hence the weak field is present near magnet's sides. Moreover, this field is directed oppositely to the one inside the magnet. You may think of this magnet as of the iron yoke in your apparatus. Yoke's field will then slightly decrease the field between permanent magnets. I bet its share will be neglectful. I wouldn't worry about that. It's quite likely that room temperature variations may influence the field between permanent magnets even more. To make your working area filled with uniform field just imitate Helmholtz coils arrangement, i.e. take two identical permanent magnets with diameters equal or bigger than the distance between their near, attracting poles. There is still a problem with "vertical" parts of the yoke, they will distort the uniformity of the field within the working area. Nevertheless, again due to the symmetry, this influence will be equal to zero in vertical symmetry plane.Following
- Mehdi Hedayati added an answer:Can I use a speed vac evaporator for drying my samples (1ml) for metabolite analysisWe have always used N2 purging to dry up the samples. However, since we do not have an automated instrument for the N2 purging, we do it manually, one sample at a time and this is becoming very laborious and time consuming as we have several samples to analyse at a time. Hence, I was thinking of changing my drying procedure and use a SpeedVac Evaporator for the same. Our SpeedVac can be operated at ambient temperatures and I have run some dummy samples and found that the temperature of my sample is actually lower than the ambient temperature because of the vacuum pressure that is generated in the chamber. Hence, I do not think there is a problem of sample heating. My run time was about 1 hour for drying up 1ml sample. I would like to know if this procedure is safe and will it create any problems if I use these samples for GC-MS or LC-MS analysis.Dear Deepti, For some reasons (e.g. Freeze drying of water insoluble products, Freeze drying of extreme water-unstable products,...) there are some solution for freeze drying organic content solution. If you have pure methanol, using collector chamber -100 C solve your problem, because methanol freeze at -98 C. but fortunately based on methanol eutectic point, in water/methanol solutions you can freeze it at around -80 C. and for second solution, you can change methanol with other organic solvent such as tBA, DMSO, DMF, Acetonitryl,...
Anyway, using nitrogen and needle is very cheap and ease method.
- Meiyappan Lakshmanan added an answer:How can I run iMM904 with Croba toolbox in Matlab?I have downloaded the txt files and now I want to import the model into the cobra toolbox in matlab. I also have libSMBL installed.Following
- Gopi G Ramkrishnan added an answer:Adding reactions to already existing mfa model - can anyone help?I need to add reactions to an already existing set of reactions used for metabolic flux analysis for a pathway. While doing that I am getting all random and unrealistic values for fluxes. What could be the probable reasons for it?Hi,
I have little experience about your problem.
I assume you are using a SBML model. If so, Antimony could help you in editing the existing file.
While editing huge models be cautious about declaration of new metabolites, their boundaries and limits.
As others pointed out, this NAD and NADH is a problem if you edit your model in matrix level.
Try using FAME. You can add new reactions online if your genome scale model is well known like Reed’s E. coli.
- George Nabin Baroi added an answer:What range of NADH/NAD ratio changes are significant if we want to increase/decrease in production of certain metabolite in a bacteria?If we want to relate changes in NADH/NAD ratio to increase/decrease in production of certain metabolite in a bacteria, then what range of ratio changes will be considered significant?For a specific condition, the redox level might not be same as with other condition. It applies same for the use of different substrate. However, the ratio is directly related with redox and it will effect the product formation. Therefore, doing a back calculation, I think you may get some idea of electron flow. that will give you an answer.Following
- Ali Salehzadeh-Yazdi added an answer:What are the ranges for enzyme concentrations, metabolites and cofactor concentrations in a microorganism?These initial concentrations are essential for me to start a simulation of a metabolic pathway (up to 10 enzymes).If you want these data for kinetic modeling, you must ask your question clearly. Nevertheless take a look at BRENDA and Biomodels databases.Following
- Sanjan tp asked a question:Adding GPR Associations?Is there an easy way to add the Gene Protein Reaction (GPR) associations to an entire reaction list of an organisms. Conventionally, one could associate the gene coding for sub-units of a protein complex, or the gene coding for isoenzymes. But manually associating the boolean logic based on protein function annotation is time consuming.Following
- Lars Freier asked a question:Flux Balance Analysis (FBA) model of plants including starch synthesis, sucrose synthesis and fatty acid synthesis.I'm looking for a Flux Balance Analysis (FBA) Model of plants including starch synthesis, sucrose synthesis and fatty acid synthesis. Need it for validating my calculated fluxes based on dMFA.Following
- Kai-Hee Huong added an answer:What are the steps in metabolic flux analysis?I am planning to apply MFA to study metabolism pathways in my bacterial strain of interest. However, I am a beginner in this field. When I study the literature, I see many reaction equations in a particular journal. I would like to ask how can we create the template (all kinds of metabolic reactions) for this particular strain?erm, i got a question. How can we say the equation is reliable if the species is different? because different bacteria act and function differently. if in the case i took the from database then it no more related to my strain but other strain...Following
- Lars Freier added an answer:Appropriated Pathway in Plants for dMFAI'm looking for a pathway in plants which can be analyzed via dMFA (metabolic steady-state, but transient isopotomic level of fractional enrichments). Has anyone a good idea?In principle yes. GCMS and LCMS was used for analyzing.
Is your paper already published?Following
About Metabolic Flux Analysis
Metabolic flux analysis (MFA) became an important tool for metabolic engineering. Right now, the Steady-State MFA (SSMFA) and the dynamic MFA (dMFA) are used for quantifying fluxes and get more information about the regulatory principles in cells. In this topic people can talk about problems and idea regarding this field.