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Why is most of my recombinant protein in the wash after His-tag IMAC?
I recently did His-tag purification for a recombinant protein under denaturing conditions. I incubated the protein extract for 1 hour. I proceeded to wash 3x and elute 2x. After running an SDS-PAGE, it was apparent that the majority of the purified protein was in the second wash (not the first or third). Any recommendations or advice?Following
What are the best known biomarkers of cancer stem cells that can be targeted by immunohistochemistry?
CD117 , MDR1 or CD44 ?
Thank you =DFollowing
Can any one help me to solve this equation using MATLAB plz?
I have a question plz
Pf = qfunc(sqrt(1+2*SNR)*qfuncinv(Pd)+SNR*(sqrt(N)))
The approximated values for Pf for different values of N are as follows:
for N=1000 Pf=0.62
for N=2000 Pf=0.53
for N=3000 Pf=0.49
for N=4000 Pf=0.44
for N=5000 Pf=0.4
for N=6000 Pf=0.39
for N=7000 Pf=0.36
for N=8000 Pf=0.35
for N=9000 Pf=0.32
for N=10000 Pf=0.3
I need to know the SNR value which satisfies the equation using MATLAB
Thanks Richard Joules. I have a similar problem. I tried your code, but the following error appears:
??? SNR_approx(i) = vpasolve((sqrt(1+2*SNR)*qfuncinv(Pd)+SNR*(sqrt(N(i)))) == qfuncinv(Pf(i)), SNR);
Error: The input character is not valid in MATLAB statements or expressions.
Could you plz solve this error?Following
What is the relationship of scan rate and surface electron transfer process on electrode?
it is an electrochemical biosensor (HRP enzyme/TiO2 film/Ti substrate).
Thank you in advance
it is an electrochemical biosensor (HRP enzyme/TiO2 film/Ti substrate)
Can you send me more information?Following
Collaboration with a Study of Long-term Serious, Human Calorie Restrictors?
I work with Drs. Joseph Dhahbi and Stephen Spindler to analyze genetic and other cell signaling patterns in long-term, serious calorie-restricted humans. The study is being done at the University of California, Riverside.
The samples were collected at the campus clinic and delivered directly to Dr. Joseph Dhahbi, the PI of the study. Separate clinical blood markers were also measured and will be reported as part of the study.
Dr. Dhahbi is willing to share some of the samples he has collected with other labs. They include one urine, two plasma, and eight cell samples per participant.
Are you interested in collaborating with this study? Please let me know and I will share more details.
Thank you for your comments, Ali. We plan diet and lifestyle to activate the cell signals, shown to extend life in both human and animal studies. So when we choose carbohydrates, I think a lot about what those carbohydrates do: whether they will help downregulate insulin/IGF-I and mTOR and whether they upregulate AMPKinase, which is central to CR benefits. There is a lot more to it of course, but getting these basic cell signals to be an enjoyable part of a daily routine is central to success with a CR lifestyle.Following
Alternatives to CF11 columns?
CF11 cellulose columns have been used to filter white blood cells out of human blood for subsequent studies on malaria parasites. Now the producer of CF11, Whatman, does no longer produce it. Does anyone has experience with alternatives? The stage distribution of the parasites should not be distorted.
I am thinking to use Whatman CF11 cellulose powder columns for purifying P. berghei parasites. I haven’t done this kind of work before. Can anyone suggest which one is better among Whatman CF11 cellulose powder columns and Plasmodipure ?
Looks like CF11 cellulose powder columns discontinued. Not very sure which one should be picked.
Any suggestions from experience user will be highly appreciated
Can someone help me with RNA purity issue?
I have been having a problem with RNA purity on my sample.
My tissue is ligament and synovial membrane, I grind the tissue using a pestle and mortar and homogenize it with Qiagenshredder.
I tried to use the QIAGEN mini kit to isolate my RNA but never got a RNA pellet so I tried to use the TRIZOL method with a glycogen carrier and got a good RNA pellet however my RNA is contaminated. I Dnase treat my RNA also with a kit from SIGMA.
My nano drop values are as bellow:
A 260= 2.1
RNA con. 84.0 ng/ul
I autoclave all my instruments, use filter tips. My bench, pipettes are clean with RNAse wipes.
Can someone help me with this?
Thank you in advance!
Thanks for the reply Sandeep,
Previously I used a gauge and syringe to homogenise and got the same problem.
After I collect the powder, I add the trizol and just straight away homogenise it. After I homogenise it, I just add the amount required of chloroform (160ul), I don't have any incubation time between that either. I centrifuge after for 5 min at high speed.
And yes I agree, I think I have to be careful when taking the aqueous phase.
Should I incubate before homogenise it? and after the chloroform step?
How can we differentiate MG 63 pre osteoblasts to osteocytes?
we are trying to differentiate MG-63 pre osteoblasts into osteocytes using the differentiaing media (containing Dexamethasone, . Sodium L - ascorbate and Beta-Glycerophosphate disodium salt hydrate) but the cells are detaching as they continuously proliferate. is their any solution for the same?
we are doing it in 12 well plate, seeding 60,000 cells in each well.
we are letting the cells grow in normal MEM media till 70% confluency, then replacing the media with the above mentioned differentiating media, and changing the media on every 2nd day. cells are suppose to stop proliferation and start proliferation when switched to differentiating media.
Sure, they have, but no valid basisFollowing
What is the difference between "Soil Water Holding Capacity" and "Field Capacity"?
Are those terms synonymous?Following
How can I calculate compliance constant values of each vibrational mode?
I am trying to calculate compliance constants for each vibrational mode. We have simulated the structures in Gaussian09 software and have been able to get the force constants in the output file (for each vibrations).
Can we directly convert the force constant of the mode to get the corresponding compliance constant by taking the inverse of the value of each vibrational mode? If not, from the whole matrix of compliance constant, how can we say which value is for which mode?
Or is there any other way to calculate the compliance constants.
We have also used Compliance 3.0.2 software to calculate the compliance constants for inter- and intramolecular bonds. But, it's difficult to get compliance constants for each of the vibrations. For intramolecular vibrational modes, the compliance constant value is changing if I choose the order of selecting the atoms differently in the software (like if I click on carbon-carbon-hydrogen-nitrogen instead of say carbon-hydrogen-nitrogen-carbon) etc. Is there any set rule how to select the atoms in the software ?
I would be obliged if you can help me regarding this.
Thank you and kind regards,
You cannot convert the force constant of the mode to get the corresponding compliance constant by taking the inverse of the value of each vibrational model. I have checked this using the values in Fig 4 and Fig 6. Chem. Soc. Rev., 2008, 37, 1558–1567. After this math, I havent got back compliance constants for the case of cyclobutane. Maybe you should ask one of the author of Chem. Soc. Rev., 2008, 37, 1558–1567 paper. In meanwhile, you can have some additional information about resonace by the Freq(internalmodes) in G09. Alternatively, you can use Atoms in Molecules analysis to identify intramolecular interactions.Following
Is anyone aware of ways to objectively measure amounts of tension in the psoas muscles?
I am planning to investigate the theory that the psoas is the "fight or flight" muscle that tends to become chronically constricted when people have post traumatic stress disorder.
Thank you, Wayne. I may have shifted the focus of my study to grounded theory on the role of the psoas in fight or flight, since I've not found any literature to back up this idea. I'll keep posting here as I move forward. I also haven't found a specific way to measure psoas activity, which would be very desirable in a quantitative study.Following
Can intuition be used as a method for acquiring information?
Can this be accepted as a bridge between the quantum and the macroscopic level by introducing new elements such as quality, degree of order, consciousness, arrow of space, and time? Interpretation of observation and experience as two different events? The selection of only one event in opposition of the possible statistics of uncertainty events?
Yes, any way of thinking brings us to new insights, but if we don't think, the brain is working by itself and it is good to trust is. I was always lucky with my physics and mathematics. Intuition is working on many different levels.Following
I need IR-spectrum of cefoperazone! Can you help me please?
I'm doing analys of cefoperazone, but i can't find its IR-spectrum! Please help me!Following
Can anyone help with my standard curve result on RT-PCR?
I have questions about standard curve for absolute quantification..For template,I have done 1:10 dilution, (1 ul template and 19 ul sterile ddH2O) in 5 points which is template concentration start from 100ng, 10ng, 1ng,0.1ng and 0.01ng. I am using Taqman method which used Taqman mastermix and custom Taqman assay (for my target gene). I've already ran 3 times for this target gene. I don't have any idea why the standard curve result has an unacceptable R2 value and do no show 100% PCR efficiency.I am using new mastermix for the third experiment. Any chance custom taqman assay caused it? Or any other aspects affecting the efficiency?. I ran the housekeeping gene 18s (the same method as this target gene), and the result is still the same as my target.
Any responds will be appreciated. Thank you :)
R2 and slope are parameters linked with different itens of your real-time PCR:
1) R2 reflects the adjastement to a linear model and is directly linked to the presence of inhibitors. With a 1:10 dilution you have to get 3.32 additional Cts. If a 1:10 diluted DNA extract has smaller Ct than the original extract, then you have strong ihnibition. Bigger Cts would reflect wrong pipetting. Among the replicates of a single extract, you should not accept Cts having differences above 0,3. You can also implement a rule of thumb to find outliers: one replicate having a Ct value 1 unit bigger than the average of the other two, can be eliminated.
2) slope of the calibration curve reflects the quality of your master mix and PCR settings. I would recommend you to take the AB 2x master mix and water necessary for both reactions (target and reference gene). Divide the mixture into two portions and add to each one the respective primers and probes. When pipetting the final misture into the tubes, use a new pipette tip for each reaction to avoid accumulation of liquid inside the tip and therefore pipetting errors. Add the DNA at the final step. Check the annealing/elongation temperature step. Usually, for taqman probes we use 60 º for 1 minute, but you have to optimize your protocol. I presume that you are using hot start taq.
The highest concentration point of your calibration curve should have a Ct value around 20-25. Check the shape of your amplification curves. Strange lines would reflect deficient amplification. If your primers are specific, I would suggest you to run some samples with SybrGreen or EvaGreen and add a melting step to the final of your real-time PCR. Primers highly specific will amplify only one fragment and you will see a perfect melting temperature pick.
You can also check the sequence of your amplicon and align it with the probe sequence. point mutations will disturbe the probe degradation and you will have deficient emission of fluorescence. Point mutations will provoke the probe depletion rather degradation.Following
In peace time and democratic normality, should the military control the police? or should the former only cooperate with the latter?
Mr. Dr. Peter Kraska,
With respect to the demilitarization of a police force, I would very much appreciate your position concerning the militarist argument of the "double use" ideology. According to this point of view, some defend that the military means should and can also be used for police activities, in peacetime and democratic normality. Is this correct?Following
How can I assign indicators to latent variables in SmartPLS?
I conducted a research to determine significant indicators of Hepatitis C among sanitary staff at hospitals. The indicators are related to demography, risk factors and organizational characteristics. I tried to do a binary logistic regression by coding all the indicators as categorical. However many of them have significant correlations among them. Thus I'm forced to use PLS. I have two questions:-
1. Can we use categorical variables in SmartPLS?
2. How should I determine the factors for PLS regression. Should I just assign the demographic indicators under one factor, risk factors under the second factor and the hospital xtics under the third? Or should I conducted Exploratory Factor Analysis? Remember that the latter might result in elimination of some of the indicators.
I hope these kinks may help
What is a suitable way to fertilized eggs gold fish?
To get the most fertilization rate and reduce the amount of fungus what methods and solutions should be used?
The best way to reduce the amount of fungus (or actually to prevent growth of fungus completely) on the fertilized eggs is to keep fertilized eggs in the methylene blue solution (normally if you buy it in the fish pet store it is 1% solution). Add 2 ml of methylene blue solution to every 10 liters of water in your fish tank where you are keeping the eggs. Keep the fertilized eggs until they are ready to hatch (usually 24 h before hatching) and than move them to a new clean tank without methylene blue. With such approach you will never ever have the fungus problem again.
Link to methylene blue is included - normally it can be bought in any larger fish pet store.Following
Early Endosome Isolation for Mouse hearts?
I've been trying to isolate out early endosomes by homogenizing heart tissues and using various subfractionation techniques to purify out cytosolic protiens, which I place in a continuous sucrose gradient (10-40%) and use western blotting to decipher their approximate location. They are always found at the 10% region, which tells me they are not breaking through the sucrose. Does anyone have any advice or suggestions for me so I can have a successful isolation them? Thank you in advance.Following
Can anybody tell me good articles about the difference between complications observed in PCI via transradial artery and femural artery?
I want articles that compare the most significant complications observed between percutaneous coronary intervention (PCI), and articles that study what complications are more related to the PCI via the radial artery and the femoral artery. Also what strategies could we (nurses) use to prevent those complications in our patients.Following
What are the latest texts, papers, or articles that describe the use of strategic alliances in business today?
My particular interest is in how strategic alliances evolve into smaller proprietary ecosystems or constellations, whereby providing that firm with a significant competitive advantage in industry. THINK: Apple and their network of collaborators.
This link may help http://www.mcser.org/journal/index.php/mjss/article/view/4316
Is Quantum Entanglement the basis for space-time and the foundation for the Theory of Everything?
NEW INSIGHT INTO UNIFICATION OF GENERAL RELATIVITY AND QUANTUM MECHANICS http://ow.ly/Nx2kI
"A collaboration of physicists and a mathematician has made a significant step toward unifying general relativity and quantum mechanics by explaining how spacetime emerges from quantum entanglement in a more fundamental theory."
"Physicists and mathematicians have long sought a Theory of Everything (ToE) that unifies general relativity and quantum mechanics. General relativity explains gravity and large-scale phenomena such as the dynamics of stars and galaxies in the universe, while quantum mechanics explains microscopic phenomena from the subatomic to molecular scales."
"The holographic principle is widely regarded as an essential feature of a successful Theory of Everything. The holographic principle states that gravity in a three-dimensional volume can be described by quantum mechanics on a two-dimensional surface surrounding the volume. In particular, the three dimensions of the volume should emerge from the two dimensions of the surface. However, understanding the precise mechanics for the emergence of the volume from the surface has been elusive."
"The paper announcing the discovery by Hirosi Ooguri, a Principal Investigator at the University of Tokyo's Kavli IPMU, with Caltech mathematician Matilde Marcolli and graduate students Jennifer Lin and Bogdan Stoica, will be published in Physical Review Letters as an Editors' Suggestion 'for the potential interest in the results presented and on the success of the paper in communicating its message, in particular to readers from other fields.'"
"Now, Ooguri and his collaborators have found that quantum entanglement is the key to solving this question. Using a quantum theory (that does not include gravity), they showed how to compute energy density, which is a source of gravitational interactions in three dimensions, using quantum entanglement data on the surface. This is analogous to diagnosing conditions inside of your body by looking at X-ray images on two-dimensional sheets. This allowed them to interpret universal properties of quantum entanglement as conditions on the energy density that should be satisfied by any consistent quantum theory of gravity, without actually explicitly including gravity in the theory. The importance of quantum entanglement has been suggested before, but its precise role in emergence of spacetime was not clear until the new paper by Ooguri and collaborators."
"Quantum entanglement is a phenomenon whereby quantum states such as spin or polarization of particles at different locations cannot be described independently. Measuring (and hence acting on) one particle must also act on the other, something that Einstein called "spooky action at distance." The work of Ooguri and collaborators shows that this quantum entanglement generates the extra dimensions of the gravitational theory."
"It was known that quantum entanglement is related to deep issues in the unification of general relativity and quantum mechanics, such as the black hole information paradox and the firewall paradox," says Hirosi Ooguri. "Our paper sheds new light on the relation between quantum entanglement and the microscopic structure of spacetime by explicit calculations. The interface between quantum gravity and information science is becoming increasingly important for both fields. I myself am collaborating with information scientists to pursue this line of research further."
Full briefing at:
I would agree gravity must emerge from energy density, which can be loosely inferred from equivalence. There are numerous ways to go about this. The holographic principle seems to be one of the least straightforward.Following
Can carbon tax be imposed like income tax for the sake of our future?
The calculus needed to drive such investment decisions is difficult - but it would be simplified considerably if carbon pricing were as common as income taxes.
This is a good question. The more abusive a country to nature, the more contributions required from it in taxes or otherwise to protect and cure and sustain nature. This puts countries to be more responsible and know that nature does not offer a free ride of exponential abuse.Following
Can cancer cells modify their phenotype without of genomic alterations ?
we consider non-genetic cell phenotype plasticity as a central process in therapy resistance. We take into account theability of cells to produce discretely distinct phenotypes, switch between them without genomic alterations and inherit the new
phenotype non-genetically across cell generations (Brock et al,2009).
Hypoxia, chronic inflammation, and redox stress are the major factors which consist of the tumor micro-environment. Cancer cells exhibit epigenetic change to survive and proliferate under those unfavorable conditions. However, it depends on whether or not the epigenetic modifications are reversible or not.Following
Do I would like to use a paired or an unpaired t test on my results?
I am investigating the value-relevance of two performance metrics. In order to test whether one metric is superior to the other in terms of indicating shareholder value creation, I ranked the total number of firms into buckets based on their performance on either one of the metrics and then ccalculated the average return in each bucket. To test the difference between these means I use a t test. The total dataset doesn't' change (also n per bucket is the same), but the buckets may differ in firm composition. What would you advice about using a paired or unpaired t test? And if I would use a paired t test, do I would like to take the correlation between both metrics over the whole data set or per bucket? Thanks in advance.
this book may help http://www.sagepub.com/upm-data/52063_00_Field_4e_SPSS_Prelims.pdfFollowing
Should I remove the ligand like molecules from active site of thrombin (1PPB) if i want to know, my ligand binding site?
1ppb is a thrombin structure that contains a inhibitor at the active site. During my literature search, I have found 1PPB was used for obtaining binding affinity informations to various ligands and its binding sites were discovered. However, They have not mentioned if they removed this inhibitor or kept it.
my aim to know at which part of molecule, my ligand is bounded so I ran docking simulations but I could not verify the information that i have read in the literature. Is this because I removed inhibitor on that molecule?
I removed that inhibitor part from 1ppb because i would like to simulate the native state since thrombin ( 1ppb) won't have that inhibitor at native state. But when i remove the inhibitor. Nearly, all of my docking simulations dock the ligand to the active site. ( even the one in the literature says it rather binds to a different site)
PS: I am using auto dock vina.
Thank your help. any information is very valuable for me.
as other say, you must remove ligand from protein structure. but this step in molecular docking is depend on software. so, program like GLIDE, GOLD, Surflex, FlexX have option for this step but the program can determine the active site, and you don't need determine active site manually.
But about program like auto dock you have two option to determine active site:
1. if you have crystallize protein structure in complex with ligand you can determine the active site by verifying the residue in active site(near the ligand) by left panel in autodock tools.
2. if you have not protein structure complex with ligand or much better to say you don't know the active site of protein, you can use blind docking or use "AutoLigand" option in Autodock tools to fine active site of protein.(read Autodock user guide)
How do I define linked and unlinked loci in single-stranded RNA viral genomes? What impact does it have on virus evolution?
In the context of RNA viruses, which represents haploid systems, how to apply concept of linked or unlinked loci?
Interesting questions - to define linked and unlinked loci in the a single stranded RNA virus is, of course, going to depended on the virus in question. The concept linked and unlinked loci comes from potential for chromosomal cross-over during meiosis, so loci closes together on a chromosome are more likely to be linked. Whereas two loci separated by a large distance on chromosomal or a region that is subject to frequent recombination are less like to be found together hence unlinked. Taking this concept into virology is a bit problematic given that the "chromosome" more or less equates to the viral genome. So essentially you would have to look at the genomes of your virus and see if there is any evidence of recombination between strains of this virus. Assuming your virus only has one genomic RNA and there is only one "hot spot" for recombination then the genes located 5' to that spot could be consider linked and the genes 3' could be considered linked as well. Whereas the two parts of the genome, either side of the hotspot could be considered unlinked. If there are multiple hotspots of recombination then a similar concept could be applied.
Impact on virus evolution - in the laboratory you might see either decreased replication or increased replication as there is no selection for fitness (typically). In the host of interest / in the field - you might see antigenic shift due to immune selection or increase in virulence due to properties such as enhanced replication or transmission. In the host of interest there is likely to be only selection for genomes that provide a competitive advantage, over the parental viruses that allow the "new" genome to become dominant in the population of genomes. These types of events are probably fairly common but the resulting viral phenotype(s) are not able to out compete the existing ones. You also need certainly conditions such as co-infection with more than one strain.
Orally disintegrating tablets, FDA and EMA BE & BA studies?
Could anybody help me with the guidances or recommendations of FDA/EMA for the administration of ODT in bioequivalence & bioavailability studies? Thanks in advanceFollowing