Analytical Biochemistry - Science topic

Ramesh K Jha Can you guide me how to prepare p-NPA solution for extracellular esterase activity assay? I read different papers, they are ether using methanol, isopropanol, or a 1:1 mixture of isopropanol and acetonitrile. Which could be the be solvent to perform this assay?

Not necessarily. The level of gene expression does not always directly correlate with the amount of protein produced. There are many factors that can affect protein expression, including post-transcriptional modifications, translation efficiency, protein stability, and degradation.

Many thanks for your reply; if I find something, I'll let you know.

Best, Jacques

Thank you, Connor Jankowski and Karoly Liliom

Hi Tom,

Apart from the data of the low concentrations, blank injections are also needed to calculate to S/N and LOD of an established analytical method.

As I am working in environmental chemistry, common we prefer to use method detection limit (MDL) to describe these things, and you can visit "https://www.epa.gov/cwa-methods/method-detection-limit-frequent-questions" for more information.

The following useful RG link is also very useful:

Saravana, it seems that fixing cells with -20 deg C methanol is recommended by more than one source.

Needs to analyze various protein, amino-acids, and enzymes. Immuno-assay techniques may be useful.

Dear Kwang

Please kindly see the following pages as they mentioned the same as yours:

1- https://www.researchgate.net/post/How-do-I-separate-essential-oil-and-wax-content-in-agarwood-oil

2- https://www.researchgate.net/post/how_do_i_separate_essential_oil_and_wax_content_in_citrus_peel_oil

3- http://www.oil-testimonials.com/essential-oils/6137/how-to-remove-candle-wax

Hope they would be useful.

Good Luck and best wishes

Hi,

Anuja Tripathi Is there any specific reason that you use the borate buffer instead of acetate?

Please obtain some training and assistance in using the HPLC system. Incorrect conclusions will be reached without years of formal training and experience. Data gathered will be invalid. Time, materials and money wasted.

Please inject a real blank (inject the same volume of mobile phase as you normally inject for the sample, but without the sample inside, just 100% mobile phase). At identical wavelengths, compare the "blank" run to a real sample analysis chromatogram (overlay the two chromatograms). If you still see the "peak", then you have a contaminated flow path (column fouling or carryover, as noted earlier).

Dear Rawaah Y. Al-sharrab,

Thank you so much for your interest. Actually, it is very difficult to solve the problem of internal standard. That is why, most of the researcher are now developing their method using external standard and to use matrix matched standard for avoiding the use of internal standard. Anyway, if you interested to work with internal standard, try to use the solvent as same as mobile phase, it may helps to minimize the problem. I read the answer made by

William Letter, he described many things, you may follow his advice. Thank you.

Hi all, I could detect pyruvate and glyoxylic acid using GC-MS.

Hi Shaun! I'm not sure where you are located, but I would recommend researching Research Experience for Undergraduates (REUs) in the U.S. Many schools here offer a summer internship program for students from another school. I am unsure of their stance on international students though.

If you are a U.S. citizen, then I highly recommend looking into internships with NASA if they align with your interests. You can learn more about those opportunities at intern.nasa.gov.

You might look up some companies that you are interested and reach out to them specifically. The easiest way to do that might be to find employees of the company on LinkedIn and reach out to them. They might be able to point you in the right direction on how to get an internship with their company.

Good luck and I hope this helps!

Hello

Really i couldn't understand why you express U/L as density

This is unit of enzyme activity per liter and usualy the enzyme activity is expressed as International Unit/ Liter, or as Katal. You can look at any enzymes book, or even general biochemistry book and read more about this, many of these books are now available free on the internet

.Good Luck

Inorganic chemistry. But..in PhD i will change to biochemistry ☺

In general, NMR is very informative and powerful technique. You can estimate the relative amount of your peptide/protein, the purity of the sample, or even the fold of the peptide/protein. You can also do interaction studies or stability tets.. Recording simple 1H spectrum can already tell you such information. But there is much more NMR can do. I would recommend to start with this very nice book:

Editor(s):

We have used this technique for one drug and confirmed stability for 12 months at 20C but it is impossible to generalise. Stability will have to be established for every individual drug and set of conditions I;m afraid

.

I suggested you to refer into the located authority dealerships of the constructive company of the apparatus namely (GCMS Shumadzu model) in the regional headquarter in your country. In addition, you could to use through the online services, the demo version and technical support of the mentioned device to troubleshoot the software or hardware.

Monika Jain Did you ever find a bacterial strain with CGA codon?

Dear Zohre Emarloo, you can use the coloremetric method using 4-aminoantipirine

Luisa María Vera , i am asking the same question as yours. i need to measure H2O2 concentration in acidified water.. have you found the answer you were aksed??? i am so glad if you would like to share here...thanks

Hi

I would have a question to your question :-). So-called ragged-ends our frequently observed when inspecting N-terminomics data. Is the case you depict with the additional Alanine removal more frequent then the cleavage of any other amino acid? do you see further proteolytic processing after the Alanine removal?

Best

Sebastian

Looks a bit like a solvent front. Have you tried an injection without the column? If you get the same shape peak (it should be much earlier then, of course) then that may be an explanation. Would then check flow-rate, length of tubing, pump performance as it seems rather late for a solvent front, although that will depend on column size and flow.

I have performed urease activity assay as per the literature (almost 6 papers) but did not get good results in terms of reproducabilty with repect to standard (thiourea). Please suggest me how to overcome the issue and also what are precautions needs to follow during estimation.

Thanks in Advance

Hi,

Please check this

This is very extensive topic to answwe. There are many papers on molten globule state by Prof. Kunihiro kuwajima and others check on pubmed.

Very interesting question

and agree with Respected Bassem Refaat

Yes, you can use spectrophotometer in measurement of OD; BUT with further disadvantages such as:

1. Time-consuming as you need to use blank before measurement OD of each sample.

2. Mistakes during procedures.

3. Difficulties in adjustment of preferable wave length.

You may try 0.1 N HCl in hexafluoroisopropanol or trifluoroethanol.

There are several other separation methods published for the detection of sorbic acid in foodstuffs. These include HPLC (Saad et al., 2005; FSIS, 2004), spectrophotometric (Campos et al., 1991), micellar electrokinetic chromatography (Boyce, 1999), and CE (Özteki̇n, 2018).

Sorry Camille Hossay ,I have dropped that project.

Mr. Thiriveedhi,

First of all, your method of choice should be mass spectrometry; because of only this method provides superior method performances up to pg.(mL)-1 concentration range; even, below.

Currently, there is used the standard procedures as they have been given in the EU directive EU Directive 2002/657/EC; https://eur-lex.europa.eu/legal-content/DE/ALL/?uri=CELEX%3A32002D0657; Official Journal of the European Communities L 221/8 , 17.8.2002.

However, you should bear in mind that the application of this directive yields to reliability of the MS analysis of complex mixtures of r = 0.98.

Convercely, the applicaton of our innovative stochastic dynamic method and formulas (Consider references [1]-[3]) improves the method performances up to 0.99996 at concentration levels pg.(mL)-1 studying mixtures of steroids. Refernce [3] shows analysis of mixtures of carbohydrates (including cyclodextrins), wherewe have achieved reliability ar r = 1 by the same (our) method.

[1]

[2] https://zenodo.org/record/3606820#.Xobg3dIzaig

[3]

Therefore, in general, your method of choice should be a MS based approach.

AOAC 998.12

Hi, you can look into my article. It is very easy.

Best regards, Matej.

Hydrolytic and oxidative enzyme production through cultivation of Pleurotus ostreatus on pulp and paper industry wastes

I now have the same problem. need software Did you ever find any?

The detector response is based on concentration C=m/V if you reduce de volume the relative concentrtion increases and thus sensitivity.

In addition to what Dr Muhammad Saad Khan said, I would suggest you consider abs values less than 0.5 for accuracy.

Also see attached papers for simple protocols to determine EE of liposomal / nanoliposomal formulations:

Colas, J. C., Shi, W., Rao, V. M., Omri, A., Mozafari, M. R., & Singh, H. (2007). Microscopical investigations of nisin-loaded nanoliposomes prepared by Mozafari method and their bacterial targeting. Micron, 38(8), 841-847.

------- and:

ElMeshad, A. N., Mortazavi, S. M., & Mozafari, M. R. (2014). Formulation and characterization of nanoliposomal 5-fluorouracil for cancer nanotherapy. Journal of liposome research, 24(1), 1-9.

K672-100 Heme Colorimetric Assay Kit

Hello, first of all, what's the sample amount in each group? In general, I suggest to test non-parametric if n < 6. For smaller sample size it is not really serious to assume parametric conditions, independently from test results. As non-parametric alternative for t-test, I suggest u-test (Mann-Whitney-Test). Best regards, Max

You could digest your PCR-product with an Endonuclease (EndoV) specific for deoxyinosine. These Enzyme is available from different companys (NEB or Thermo).

Isolation

- Principle

Nicotine is a classical alkaloid the separation of which teaches the

main principles of an alkaloid isolation. Consisting of a combination

of two tertiary amines of which the pyrrolidine is the stronger basic

one, nicotine is protonated in the plant and forms carboxylate salts such as formate, acetate or maleate. Therefore, the fi rst and typical step is to bring the alkaloid into a distinctively strong alkaline environment, such as NaOH solution to cleave the organic salts and release free nicotine into the aqueous solution. Nicotine is readily soluble in water due to its ability to act as an acceptor for hydrogen bonds to water.

Another useful property is its volatility with water vapour. This allows

steam distillation to be used for a very selective separation of nicotine

from many other water-soluble tobacco constituents. In the distillate,

the alkaloid is protonated by addition of hydrochloric acid and the

nicotinium ions formed are precipitated by addition of sodium picrate

solution. The yellow nicotinium picrate formed is pure. To obtain the

free alkaloid base, a second alkaline cleavage as with the starting tobacco is necessary with the nicotinium picrate. The free base is extracted from the basic solution with diethyl ether and fi nally purifi ed by a distillation in vacuo.

Hello, see the info below you can find some news there.

Good luck!

The most precise way to determine \bar{\nu} for small volumes and molar concentrations is to measure the natural frequencies of a U-shaped tube filled with blank buffer and protein solution (see doi:10.1016/S0006-3495(98)77735-5).

I think the intention is for the final SDS concentration in the samples to be analyzed to be 2%. Since adding the SDS from a stock solution (typically, 10% w/v) slightly dilutes the samples, you should account for this dilution after the protein measurement is made.

For example, if you add 25 µl of 10% SDS to a 100 µl sample to get 2% SDS, the sample has been diluted to 4/5 of its original concentration. Therefore, after the assay is done, you should multiply the protein concentration in the original samples by 5/4. However...

The standards used for the BCA assay (usually bovine serum albumin) should contain the same amount of SDS as the samples. If you treat them the same way as the samples, that is by adding the SDS to them after they have been prepared at a range of concentrations, then you don't have to do the correction stated above, because it will have been incorporated already into the standards.

I have results of UV spectrum as follow:

Abs 370= 3.34

Abs 370 (blank)= 0.069

Abs 280= 2.64

if the coefficient is 22000,

How can I calculate carbonyl content??

Electrodes should always be in touch with the (buffered) liquids or else there will be no current possible, hence no electrophoretic movement. You may use a filter or semipermiable device to separate the electrode conpartment from the electrophoresis buffer. Take care that the buffer in the electrode compartment is exact the same as in the adjacent separation compartment.

Check commercial equipment how your punp could be isolated.

Analytical Biochemistry

Book by David Holme

Arushdeep.. X-ray diffraction, XRD, is the major tool for the examination of crystalline solids. Powder XRD may furnish the preliminary data on the crystallinity. The more precise technique is the XRD examination of a single crystal of your material. You may get after using the supplied software, the bond lengths and the exact distribution of atoms within the crystal. Good luck

Check the reaction conditions, such as anhydrous and anaerobic. Use NMR，chromatography and other methods to check whether the impurities and whether they are the substances you need.

You can collect urine and serum samples and make the Comparison between them results.

The best extraction method depends on the finished product you want to obtain. As a general lab test to get good quality and purity I suggest to go for QWET extraction by using lab/food grade 200 proof ethanol (NOT Denatured). The starting material should be dry (humidity<10%) and fine-milled (1mm). The ethanol shall be cold as well in order to minimize the presence of polar compounds such as chlorophyll in the extract. At a temperature around -30°C you should be able to maximise the cannabinoids (non-polar) and cannabinoid-acids (polar) yield while minimizing the undesirable compounds (chlorophyll, waxes ...) that will be kept frozen in the plant material. Dry-ice crumbs in ethanol will help obtaining a quite effective slurry, while a vacuum chamber and/or gentle stirring will accelerate the process reducing the extraction time to approx. 15-20min. so prepare the cold ethanol with a lab freezer or simply mix some dry-ice and ethanol in a separate container: ethanol shall be at least in a 4:1 ratio with the green matter. In a stainless steel container mix the milled plant material with dry-ice crumbs, stir and let it rest for 10 min, then add cold ethanol and gently stir during 15-20min (don't wait until dry-ice is completely dissolved or undesirables will defreeze and be extracted). The resulting tincture shall be then well filtered (25µm mesh or smaller) to avoid plant particles. If you need a very clear oil, let the tincture rest in freezer (-18°C) for 12 hours and remove the lipids collecting on the surface (winterization). As last step evaporate the ethanol at low temperature to minimize the loss of highly-volatile terpenoids (at 20°C) or at moderate temperature (40+°C) if you don't mind losing some of them.

You can use evaporate the mixture under reduced pressure by a rotavapor

There are 48 wells in Elisa technique for small samples like in animal researches.

The strength of a given interaction can be judged through the association constant K or the dissociation constant Kd. Very roughly, and taking 1 M as the reference standard state concentration:

- Low affinity: Kd larger than 10^-4 (> 100 microM)

- Moderate affinity: Kd between 10^-4 and 10^-7 (100 microM - 100 nM)

- Low affinity: Kd smaller than 10^-7 (< 100 microM)

However, people working on in different fields may have different considerations. Thus, a Kd of 10^-6 (1 microM) can be considered as high affinity in metabolism regulation, while it can be considered a low affinity in antibody design.

And this is related to another way to judge the strength of an interaction which takes into account the potential concentrations of the interacting molecules. Then, a Kd of 10^-6 (1 microM) has different physiological consequences if the interacting molecules are present at mM (complex predominates), microM (complex and free molecules at comparable molar fractions) or nM (free molecules predominate) concentrations.

Dear Nikhat, every time an adenylate is added to the chain, a pyrophosphate is released, so there must be as many pyrophosphates as there are bonds formed between nucleotides. This is summarized as follows:

2A -> A-A + PP

3A -> A-A-A + 2PP

4A -> A-A-A-A + 3PP

5A -> A-A-A-A-A + 4PP and so on

It can be generalized that the number of oligoA (oA_n) chains is

Where n is the number of A present in the oligoA chain. In your case, you want to know how many moles AAAA were formed on average, you should do the following operation:

Postscript: if the oligomerizing enzyme removes all the pyrophosphates from the first adenilation step (2A -> AA + 2PP) then the correct calculation is the one signaled by Adam B Shapiro . Could you tell us if the enzyme removes PP from the first adenylate? This detail is important in small chains but practically irrelevant in very long polyA chains.

I trapped nitric oxide and I've got many remarks and observations.

If you have Fenton-like reaction, who knows, as the solvent is determining what you get. Otherwise, are you 100% sure that you are detecting what you don't expect? Check DMPO with Fenton, see if it's good. If you are using the parameters from literature but your solvent is unusual - those can vary. Also possible, you are simultaneously having DMPO-OH and DMPO-OOH. This is the known drawback of DMPO - use DEPMPO

And you can check my blog with more Q&A related to DMPO:

Following

what is the interpreation of increaseing of decreasing metabolic activty?

Lorraine,

I think this paper is useful for you to pyruvate decarboxlase assay.

Thanks a lot

Try dragendorff's reagent and vanillin in sulfuric acid.

Hi Navid,

Thanks for your interest! I went ahead with the C18 and it worked. The mass spectra looked fine, we identified our proteins without any difficulty and there wasn't any effect on our mass spectrometer so looks like the C18 removed any glycerol remaining in my sample.

@ Pascal Dubé @ J.D. Franco-Navarro

Yes SPE on N-vinylpyrrolidone-divinylbenzene copolymer would also work nicely, and you can find commercially prepacked cartirdges.

hi Acrot,

here you can buy it

You can always calculate the pKa(s) with quantum chemistry method. Here is an improved model for your reference,

If you are trying to polish purified BSA by removing fatty acids, you can run it over a cationic resin.

There are many papers on the subject of mycobacterial drug targets. Here are a few of them.

Sounds like som contamination with phenol but since you should do DNase digestion, precipitation, washing, reconstitution and cDNA-Synthesis, your problem is likely to be gone afterwards. Just continue...

Dear Sir,

I know about very little bit for your helping purpose i am sharing with you this links please refer might be helpful to you for getting some idea about manufacturing of high temperature and high pressure reference electrode.

https://open.library.ubc.ca/media/download/pdf/24/1.0071276/2

regards

Hi Anna,

I have “P6887 SIGMA Pepsin from porcine gastric mucosa lyophilized powder, 3,200-4,500 units/mg protein” Do you know how can I make the stock? I will be very great full if you help me.

Thank you so much

Try using speedvac concentrator or use volatil salts like ammonium carbonate

For metal determination in animal tissue, you must use a very energetic digestion; usually HNO3 and H2O2 mixture provide the lowest backgrounds for atomic spectrometry. With these reactants, you need temperatures above 200ºC for about 10 min. This digestion scheme provide lipids break and lower interferences.

Follow the original formulation of G. Dana Johnson [JACS 73 (1951), 5888]:

"To make one liter of approximately 0.25 M reagent, 50 g. of 2,4-dinitrophenylhydrazine was dissolved in 600 ml. of 85% phosphoric acid in a one-liter beaker on a steam-bath. The solution was diluted with 395 ml. of 95% alcohol and clarified by suction filtration"

If you are using DNPH that is supplied with a minimum wet weight (for safety reasons), adjust the mass of DNPH accordingly. eg. If supplied as minimum 33% wet weight, use 75g wet DNPH in the above formulation.