Reagents - Science topic

How big is your HRM amplicon? And what are you targetting? A single SNP? Methylation?? From the melting curves, it looks like your amplicons might be quite large ??

Yes sure

We stain the roots with hematoxyllin (this is mitosis). Everything OK with cell walls. (Dissolve 1 gram of hematoxylin powder in 50 µl of distilled water and leave for 3 days. Then add 50 µl of glacial acetic acid and hold the iron clip for a couple of minutes). Boil the roots in hematoxylin over an alcohol lamp.

Hello Sadia Haque Khan,

Even if you had the opportunity to work along with members experienced in cell culture, since you are new to cell culture, it will take a while till you develop the skills of handling cultures.

Keep following the sterile practices learnt from your seniors. Observe your seniors when they handle cultures. You should record even the minute details such as the way they open the knob of the culture flask during feeding the cultures or subculturing. For instance, the knob of the culture flask should be kept in such a way that the inner side of the knob faces upwards and does not touch the surface of the laminar hood. The pipet tip should not touch any non-sterile surface, and so on and so forth. You will be able to master these skills with practice.

Till you master these skills, I suggest you add antibiotics to the growth media which will help prevent contamination. Generally, antibiotics are not supposed to be added to cultures. Once you get the confidence of handling cultures without any contamination, you may discontinue the use of antibiotics.

Good Luck!

Salts of palladium and gold I think,

and Inorganic or metallic compounds typically do not produce significant color changes with the Marquis reagent

Hello Ambra.

Ellman's reagent (DTNB) reacts with any molecule that has a free thiol group (-SH), generating TNB (increasing yellow color), with a gain in absorbance that can be read at 412 nm. Due to the non-specificity of DTNB with the different thiols in your sample extracts (cysteine, GSH, coenzyme A), it is necessary to evaluate glutathione concentrations using an exogenous GR. Depending on the concentration of glutathione in your sample extract, you will obtain a proportional slope (delta A/delta T) on the spectrophotometer. Therefore, in addition to analyzing your samples, it will also be important for you to perform a calibration curve using exogenous glutathione (analytical standard), so that you can later interpolate your data obtained with biological samples using this calibration curve. Answering your questions, the reason why NADPH is the last reagent to be added is precisely because before it is added, all the GSH in your sample will be in the form of GSSG (as it has already been completely oxidized by DTNB). When adding NADPH, GR will begin to convert it into GSH, which then reacts with DTNB until all this NADPH is exhausted, thus generating a slope proportional to the concentrations of its glutathione, as mentioned previously. To be able to differentiate the initial GSH from the initial GSSG, you must perform a new test using vinylpyridine. Vinylpyridine has the ability to complex with the initial GSH, leaving only the GSSG available for you to carry out the cyclic reaction with GR, NADPH and DTNB again. This way, by calculating the concentrations of total glutathione and GSSG, you can determine the initial GSH concentration: Total glutathione = GSH + GSSG. It is important to remember that each GSSG is equivalent to 2 GSH. If you have any specific questions, you can ask them in the comments below.

Best Regards.

I have earlier faced issues of this sort. I still am facing it while performing S1 nuclease DNA fiber variant.

My advisor suggested dilution with unlabelled cells. I tried to dilute the labelled cells with unlabelled cells. It has given me good results with the normal fibre experiments (except S1 nuclease variant, where nuclei tend to clump). You will get better spreads and data if you follow this. Hope this helps.

I would also appreciate if anyone could let me know tricks with the S1 nuclease experiment.

Ganesh Pai B

NaI can be replaced by KI, since iodide reduce Tm of protein and cause instability of folded protein, which had its purpose for protein denaturation, but potassium may increase the pH. For sodium N-lauroylsarcosine, as long as sodium lauroyl sulfate provide a membrane disruption ability, then the DNA should be released as the same way as original protocol. All your reagents should be in tris-HCl and adjusting to a final pH 8, because in this pH tris has its best buffer ability.

Best

There are 2 main reasons why the protein could be precipitating: electrostatic interactions or hydrophobic interactions.

Increasing the salt concentration would probably overcome electrostatic interactions. You would have to remove the salt later, which is easy to do by passing the column through a desalting column or by dialysis. Changing the pH by several units could also help, but you have explained why this is a less desirable approach for you.

In my opinion, the more likely reason for the insolubility is hydrophobic interactions, since the interior parts of proteins are hydrophobic. You could probably keep the protein in solution using a detergent, but removing detergent later can be troublesome. There are a few detergents that are compatible with mass spec, however. The other approach would be to add a strong chaotrope, either urea or guanidine-HCl, at several molar. These are small molecules that can be removed later. You would have to experiment to find the minimum necessary concentration to keep the Zn²⁺-free protein in solution, especially if you don't want to completely unfold the protein, since refolding a completely denatured protein can be a challenge.

I have the same problem. Please advice

On looking Slide at first instance , it’s is not typical rod cell myocytes. Might be the fault at isolation , proccessing. Better to repeat the sampling

Michelle Darmawan

You can try to achieve this pH by increasing the temperature, pressure, or adding another extract.

Michelle Darmawan

Silver ions are well reduced at room temperature. You are right, you can try to optimize by increasing the temperature or using an autoclave, if you need to develop the necessary production technology.

I would start by amplifying a common housekeeping gene to check that there is cdna and that it can be amplified. You can waste a lot of time worrying about the pcr conditions when actually the problem may be at the template part of the process. Then I would start with a low annealing temperature (8c below the melting temperature) and too many cycles (36) because it is easier to clean up a working but dirty pcr than to guess where the problem might be when there is no product, Check if the template sequence is very high GC or AT in which case secondary structure may be a problem and a high GC buffer ( betaine plus dmso) may help and also check if any known snpd exist under the 3' ends of either primer sequence in your species. If the primers are old and inherited from another researcher they may be degraded so try amplifying a gdna sample just to prove that the primers can work if the product is not too large

Proteins don't always run at the expected molecular weight on SDS-PAGE. I'd suggest sending a sample of the mostly purified protein off to a mass spec lab to determine the molecular weight of the whole protein.

Meanwhile, it wouldn't hurt to include protease inhibitors when you lyse the cells. There might be a protease-sensitive site near the N- or C-terminus.

Sorry I didn't. We're going to draw down our last stocks of it soon. Then we will have no choice but to hire a full time biochemist to make it... I'll be sure to check back into this thread, if we're ever successful with that.

In such cases, it is preferable to prepare it daily and avoid its instability as a result of it being affected by various conditions such as light, temperature, etc. It is best to prepare the amount as needed daily

Aromatic nitriles are versatile intermediates in organic chemistry, serving as precursors to amines, amides, and carboxylic acids among other functional groups. The selective reduction of these nitriles to primary amines in the presence of other functional groups like OH requires careful consideration of reagents and conditions to achieve high selectivity and yield. The challenge is compounded by the resonance stabilization of the nitrile group when attached to an aromatic system, which can impede reduction. Additionally, the presence of an OH group introduces the risk of over-reduction or side reactions, necessitating a strategy that can accommodate both functionalities without necessitating protection of the OH group.

Methodologies for Reduction

1. Catalytic Hydrogenation

Catalytic hydrogenation using hydrogen gas (H₂) and a palladium catalyst (Pd/C) is a widely adopted method for reducing nitriles to primary amines. However, the effectiveness of this method in the presence of an OH group and an aromatic nitrile can vary based on the substrate and catalyst used. Palladium on carbon (Pd/C) under mild conditions can offer a route to selectively reduce the nitrile without over-reducing the OH group, though the reaction may require optimization of pressure and temperature to achieve the desired selectivity (Rylander, 1979).

2. Chemoselective Reduction Agents

Selective reduction agents such as Nickel Boride (Ni2B), formulated by the in situ reaction of Nickel Chloride (NiCl₂) with Sodium Borohydride (NaBH₄), have shown promise in reducing nitriles to amines while preserving other functional groups like OH (Girard et al., 1998). This method leverages the chemoselectivity of nickel boride for the nitrile functionality, potentially offering a viable pathway for selective reduction in complex molecules.

3. Transfer Hydrogenation

Transfer hydrogenation represents an alternative strategy, using formic acid or ammonium formate as hydrogen donors in the presence of a suitable catalyst, such as Ru, Rh, or Ir complexes. This approach has been successful in reducing aromatic nitriles to primary amines under relatively mild conditions, with the potential for high selectivity (Casey et al., 2007). The compatibility of transfer hydrogenation with various functional groups, including OH, makes it an appealing option for selective reductions.

4. Avoidance of Protective Groups

The strategic avoidance of protective groups is a guiding principle in the design of reduction methodologies. While protective group strategies can offer a route to selective reductions, they introduce additional synthetic steps, increasing the complexity and time required for the synthesis. The methods outlined above represent approaches that, in principle, obviate the need for protecting the OH group, thereby streamlining the synthetic process.

References

Flex-T analysis is a method for identifying and characterizing antigen-specific T cells. Here's a breakdown of the process:

Important Note: Flex-T analysis is a complex technique requiring expertise in cell culture, flow cytometry, and immunological assays.

I would start by guessing that it is 1X, since no other information is available. You could try using the method given in the original paper by Bradford. https://hoffman.cm.utexas.edu/courses/bradford_assay.pdf

I use a microplate version, in which a 10 µl sample in water or a non-interfering buffer is mixed with 200 µl of 1X Bradford reagent, and read at 595 nm after 5-10 minutes. The amounts of BSA depend on the quality of the Bradford reagent, but I use either 0.5-5.5 µg or 1-11 µg to make the standard curve. I fit the absorbance vs. µg BSA data with a 2nd order polynomial, but a linear fit may work well, especially for the lower concentration range.

One possibility is that the amount of protein added is too high. Try diluting the sample with water and doing the assay again.

Another possibility is that the amount of other substances besides protein is the problem. This could be nucleic acids and fats, for example. Some additional preparation of the sample may be needed.

Thank you very much B. C. Menarim !

We are taking your suggestion

Adam B Shapiro thanks this looks very helpful for me

The spectrum of the Bradford reagent is affected by dilution. This is probably why you saw the effect of water. When performing the Bradford assay, it is important to make sure that all samples have the same volume, so that the dilution of the Bradford reagent is the same in all cases.

The question is: what are the contaminants in the less pure species and are their compositions reproducible? Generally, I would suggest to make the first experiment with a small batch of the highest purity and then see if the lower purity also does the job with the perspective for upscaling, but if the contaminants are not reproducible, the whole coating quality probably won't be.

The BCA assay is not compatible with reducing agents. If there is dithiothreitol, mercaptoethanol, or other reducing agent in your samples, it will interfere. See the product literature for a compatibility table.

The blank should ideally be of the same exact composition as the sample, so it should include RIPA(X2) and protease inhibitors in the same proportion as the samples.

Hi Akshatha Banadka , did you get the protocols? If yes, could share with me, please?

Industrial chemistry often uses immobilized enzymes for synthesis. See https://en.wikipedia.org/wiki/Immobilized_enzyme.

Hi Wanji Li

Did you find another product? I'm on the same search!

I found this at Sigma: Product R1753 (https://www.sigmaaldrich.com/DE/de/product/sigma/r1753)

Thinking about ordering it, although the description makes me feel uncertain.

Best wishes!

Christina

Dear Akanksha Vyas

You could adding NaCl (100-250 mM) to the culture medium and/or modify agitation speed.

Good luck

Maura

If the protein is in the insoluble fraction because it did not fold properly, which often happens, then it will probably be necessary to completely denature it with concentrated urea or guanidine-HCl, then refold it using any of several techniques. As this process can be difficult to achieve without several experiments, it is often worth spending some time to try to avoid the problem in the first place.

Sometimes, a portion of the expressed protein can remain in the soluble fraction if the expression conditions and/or the expression strain are changed. One approach that sometimes works is to lower the expression temperature. as low as 16^oC. This slows down expression, possibly allowing time for the some of the protein to fold properly instead of aggregating.

I have a protocol for mouse brain tissue and NR2A. Would this work for you?

The most commonly used transfer buffer is Towbin's (doi:10.1073/pnas.76.9.4350, in effect SDS-PAGE running buffer with both SDS to increase the mobility of proteins and methanol to reduce it). doi:10.1006/abio.1993.1313 uses separate buffers on the membrane and gel side to resolve this conflict. In my hands, however, Dunn's buffer (10 mM NaHCO₃, 3 mM Na₂CO₃, doi:10.1016/0003-2697(86)90207-1) is cheaper and works better.

I also find that tank blotters work better than semi-dry, probably because the Joulean heat produced by the current is distributed into a larger volume.

Hello Melle Katerberg

I agree with Julie Ann Dougherty . RNA quality can be affected by impurities like phenol which can be carried over to samples. Residual phenol is assumed to inhibit PCR reactions by denaturation of enzymes such as polymerases and reverse transcriptase in a concentration dependent manner. So, try to use lower concentration of RNA than what you would be presently using.

You may want to refer to the articles attached below for more information.

Best.

No, I haven't. But it is logically sound.

I would just use ~40% more µg of the 14kb plasmid, compared to the 10kb version....

thank you so much!

YES, you can, just before starting you have to add a step of RT deactivation (e.g. 94°C for 10 min) at the beginning of your PCR thermoprofile. However, the cost of the run will be more expensive when using RT-mastermix than ordinary PCR-mastermix.

I've never done this, but I can think of a pretty easy way to do it, if you are content to measure the concentration of phosphate ion (PO₄^3-), or phosphate+pyrophosphate, in the medium.

At various time points, centrifuge the culture to pellet bacteria and remaining insoluble phosphate. Prepare serial dilutions of the supernatant in water and measure the concentration of phosphate ion using any of the many versions of colorimetric molybdate-based phosphate detection reagents (easily prepared or purchased).

To also measure the concentration of pyrophosphate, precede the measurement step by including the enzyme inorganic pyrophosphatase (commercially available) to convert pyrophosphate ion to 2 phosphate ions.

If you are also interested in measuring organic phosphate, it requires first treating the supernatant with hot sulfuric acid and peroxide to convert organic phosphates into inorganic phosphate ion. This is a rather nasty procedure, best avoided if unnecessary for your purpose.

i suggest you use XRF for determination of cobalt and for cadmium you can use FTIR and raman az well. but depends on the condition of their bonds in molecule they need diferent methods

Thank you

The effect of NaOH is probably due to alkaline hydrolysis of the bond linking the fluorophore to the rest of the fluorogenic reagent, generating the fluorescent product.

If it is labeled as 1000X, it is intended to be added at 1 part per 1000 by volume. There is no way to convert to molarity unless the concentration in molarity or g/L is stated on the package or in the product information.

You can prepare the Liposomes with the active compound and the nanoparticles by ultrasonication or extrusion, and add the polymer during annealing.

Or if you first encapsulate the active compound with PGLA, form Liposomes by extrusion integrating np and PGLA

Did you check or replace the liner and septa with the new ones? Is the liner inert and clean and septa is robust?

Since you indicated the solvent and system blanks are okay maybe interrogating the MS spectrum using peak annotation of each peak can indicate whether it is a column bleed derived, liner, or septa issue. Each mass (peak) gives clues about the problem...

On the other hand, BSTFA derivatizations are not very compatible with aqueous mediums. Hydrogen bonding affinity presented by alcohols and water can disrupt the desired reaction. Therefore it is advisable to perform a reaction in a hexane-like hydrophobic solvent environment. Otherwise inefficient reaction yield and unwanted impurities could be observed.

Hello R. Saxena , I hope you got your microstructure.

I am also working on 17-4 PH SS could you please help me in suggesting etchant with time for microstructure revelation of 17-4 PH SS, I am also facing same problem.

I've never tried to anneal oligos without salts. You can easily make appropriate buffer in-house by just searching for "annealing buffer". Good luck!

Dear colleagues,

The question was trivial.

ByBoP is a form of HOBt.

We have try it and it is working.

I hope that will be helpful for other researchers in SPPS.

Kind regards,

Robert Gradinaru

Chemistry, UAIC, Romania

Paulina Troncoso Hola! You need to know or measure the particle size distribution to be able to calculate the number of particles/mL in your system.

There are several chemical reagents commonly used to detect phytochemical constituents on TLC (Thin Layer Chromatography) plates. The choice of reagent depends on the type of compounds being analyzed and the specific functional groups or classes of compounds being targeted. Here are some commonly used spray reagents for TLC analysis of phytochemical constituents:

It's important to note that the choice of spray reagent depends on the specific analytes being targeted and the desired detection sensitivity. Additionally, it's recommended to use appropriate safety measures and handle these reagents with care as some of them can be hazardous. It's always a good practice to consult

Dear Dr Vijay Arjun Bagul

Since my lab have no OPA and the order is still in process, thus, I came up with the idea to find another possibility to analyze SMM without OPA.

I found several references that analyzed SMM without OPA, I followed those papers but I'm still failed.

Hello Adriana Quecho

I am keeping quite well. Hope you are well too.

You could succeed in making RNase decontamination reagent in the laboratory. Follow the link provided below.

https://pipettejockey.com/2016/05/06/make-your-own-nucleasenucleic-acid-decontaminating-solution/#:~:text=For%20RNase%20Away%20several%20SDSs,sodium%20lauryl%20sulfate%20(SLS).

Best.

There several possibilities:

There are a few potential sources of contamination to consider in your viral titration experiment:

Hi Huang,

Lipofectamine 3000 reagent certainly worth trying if you're facing low transfection efficiency with HEp-2 cells. Always perform control experiments and assess cell viability and transfection efficiency to ensure that the new reagent is working effectively for your specific application. Additionally, consider running parallel experiments with your previous reagents for comparison.

Transfection can be a trial-and-error process, and the right reagent and conditions can vary depending on the cell type and the specific plasmid being used.

of course you can calculate conversion of yor process. Related of your second question, you have to be in mind that software respond to a set of conditions made by THE OPERATOR, At final of the simulation software is not responsible by the results. This responsability is of the operator, that, at the end has to check if the response is resonable ( for example, if there is a strange current composition, etc). So the type of reactor you should use depends of your reaction and the set of conditions you choose. I recommend to visit software manuals and some literature to see what kind of reactor people are choosing for the type of reaction you intend to use. Also I recommend you to check in the literature what kind of themodynamic model you will choose: it may influence your results.

in other words, simulation is a very and fast tool to study process, but you have to be sure what you want to sudy and set the themodynamical model and in the case the type of reactor to be helped by software.

Run a PCR with 2 vials each of no primer, no-template, and master mix alone. And in case you are using strips, better do it in vials so that contamination can be avoided.

Niti Sharma Thank you so much ma'am

I got it ma'am and i got by this method same what i got from form this excel f (=TREND(G158:G163,H158:H163,E170)) which is 20.3 ug/ml But ma'am How can i got TheTotal Phenolic Content TPC mg/g.

The only components you can vary are the amount of cDNA and primer concentration.

For everything else, follow the instructions in the kit since those are already optimized (e.g. temps to use for polymerase).

Amount of cDNA is going to depend on expression of your gene-of-interest so you'll have to figure it out by trial and error.

Good luck!

Hi there, this is the reason this kit (see below) was invested. It is compatible with almost any buffer.

I hope it helps!

Hello Aagosh Karhale

The Fc part of the antibody molecules interact with a plethora of immune cells such as B lymphocytes, monocytes, macrophages, including granulocytes. These cells express a variety of Fc receptors on their surface and can therefore generate increased background or even false positive signals in flow cytometry analysis.

This unwanted antibody binding to Fc receptors can be avoided by blocking, which is done by saturating the receptors prior to staining the cells with labeled antibodies. The potential blocking reagents that could be used include specific anti-Fc receptor antibodies or excess purified IgG, or excess (unpurified) IgG in the form of adult serum.

In theory, using antibodies against Fc receptors is the best way to eliminate unwanted signals mediated by Fc receptors binding, but you may not always have a well-optimized antibody pool against Fc receptors.

I would recommend the use of purified IgG to specifically block Fc receptors.

You could use 1 μg of IgG per 1 x 10^5 cells for 15 minutes at room temperature prior to staining. This will reduce unwanted Fc receptor interactions. When performing Fc receptor blocking with IgG while using unconjugated antibodies, it is imperative that the IgG for Fc receptor blocking not be of the same species of origin as the primary antibody to prevent the secondary antibody from recognizing any Fc receptor bound IgG.

You could also use a blocking solution prepared by adding healthy human serum to PBS to a final concentration of 10%.

Best.

Try this reference if you want to DIY the method ( ). In general you add 10x the mass of PVA than your malachite green.

Thanks for your suggestions

Dear Manuele,

Many thanks for your kind suggestions and the link.

Yours,

Siavoush

Dear Collince Omondi Awere,

Various grades of BioPBS were dissolved in one of the three solutions: chloroform, a cosolvent system of chloroform/N,N-dimethylformamide (DMF), or chloroform/dimethyl sulfoxide (DMSO).

See: Electrospinning Process and Structure Relationship of Biobased Poly(butylene succinate) for Nanoporous Fibers, 2018, DOI: 10.1021/acsomega.8b00332

Best wishes,

Ali

Dominique Liger Nick Gee Thank you both! I replaced the premix with one at pH 8 and removed the EDTA, and now can see colour change within in the 250uM - 5mM range.

Hi Kiriakos,

I have done RNA isolation from different types of tissue i(n the time when no RNA isolation kits were available) with the method described by Chomczynski and Sacchi, 1987 (attached). I am not sure if the purchase of all components required is cheeper than a comercial kit but this might be dependent on the number of samples you need to process.

Best,

Murat

The method from https://nrl.northumbria.ac.uk/id/eprint/48184/1/wright.jennifer_phd_16029289.please (page 83)

Wolfgang Schechinger Finally, i got the 150 linearity with single point calibration. Thanks for your reply.

Thanks Wolfgang, I appreciate the detailed answer!

I have gotten somewhat useful results out of this once, in that I could at least identify viral capsids in infected cells, but it obviously depends on what information you exactly need for your analysis.

The treatment is harsh and thus the ultrastructural preservation is ... bad. Expect the lipids to be pretty much gone, and the protease treatment doesn't help either ;)