Assays - Science topic

You can use QuickExtract DNA Extraction Solution (LUCIGEN) and follow manufacturer's instructions.

Hello Malcolm Nobre

Thank you very much.

Instead of using falcon tubes, you can use microcentrifuge tubes that fit into a standard microcentrifuge. These tubes typically have smaller capacities (e.g., 1.5 mL or 2 mL), but they are suitable for processing smaller volumes of sample.

Here are a few examples of commercially available SOD assay kits:

These kits typically come with detailed protocols and all the necessary reagents for performing the assay.

PBMC don't turn black. You have a very strange problem. Either your microscope is damaged, or a very weird molecule got into your culture somehow. How much cobalt is that?

PBMC do largely die over time, so it's difficult to estimate what portion might be alive based on other people's experiments. Can you access a Hoffman's contrast microscope? With that, you could discriminate live from dead PBMCs without any staining, by just looking at them. The live ones look nice and spherical, and the dead ones flat and kind of crinkled.

You could also use a dye like MTS to tell if there are any live cells at all in your strange culture.

Dear Kais Khudhair al Hadrawi

thanks for your kind and thorough reply.

In RBC, I am actually looking for proteins to be used as biomarkers for diseasese not related to RBCs. Therefore, RBCs for me are just a biological matrix, and the proteins I am going to investigate should be not related to RBC function. I can consider my sample as sufficiently pure, since I careflly wash it many times to remove serum and carefully remove the white ring of leukocytes. The reason why I would like to normalize for haemoglobin content instead of total protein is because, as you mentioned, it is simpler, faster, more standardized and requires far less amount of sample.

You can refer to this paper. There is quite an extensive discussion regarding TFC assay.

If you have pure proteins in solution, you can use any of several protein assay kits that are commercially available (examples, BCA assay and Bradford assay). Each one requires a standard. The standard, in these cases, can be the same protein that you are measuring, since they can be purchased.

If the pure proteins are in a solution in which there is nothing else that absorbs in the far UV, you can use the method of Gill and von Hippel to calculate the molar extinction coefficient (or look it up somewhere), and measure the UV absorbance to get the concentration. This method requires a solution of 8 M guanidine-HCl to fully denature the protein.

Gill, S.C. and von Hippel, P.H. (1989). Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182:319-26

The refractive index of the solution of pure proteins can be used to measure the concentration of the protein, using the refractive index increment (dn/dc) for globular proteins (0.185 mL·g⁻¹), if a refractometer is available.

For pure proteins, you can send them to a specialist lab to measure the concentrations by amino acid analysis.

If the proteins are present in mixtures, then you can use an ELISA kit with specificity to the protein of interest. These can be purchased, but are expensive. Standards of the same proteins of known concentration are required, and should be supplied with the kit.

If the protein of interest is a major component in a mixture, a semi-quantitative method would be to use SDS-PAGE, Coomassie Blue staining, and densitometry to measure the percentage of stain in the band corresponding to the protein of interest, and compare that to the total protein concentration measured by some other method, such as Bradford or BCA. This requires a densitometer or gel imager with this capability.

There are a variety of other possibilities, depending on the nature of the sample, the availability of a standard of comparison, and the available equipment.

Hi Giorgia Martelli

I have used Quercetin and Ascorbic acid which effectively quench the relevant ROS release in DHE.

Hope it helps,

Thanks,

It is important to also consider that the availability of oxygen to the outer layer of cells is different to what the inner cells in the 3D culture are receiving.

In miRNA TaqMan assays, exogenous control standardization is crucial for accurate and reliable quantification of microRNA (miRNA) expression levels. Exogenous controls, also known as spike-in controls, are synthetic RNA molecules that are added to each sample at a known concentration before the RNA isolation step. These controls serve as reference points to normalize variations in sample preparation, RNA isolation efficiency, and reverse transcription efficiency across different samples.

Thank you for the answer! I'll review my protocol again.

Thank you

Kais Khudhair al Hadrawi Phan Thi Lam Hong thanks very helpful

Thank you for the explanation@ Kais Khudhair al Hadrawi.

Fluorescence Recovery After Photobleaching (FRAP) is a technique used in cell biology and microscopy to study the dynamics of molecules within living cells. Here's how it works:

FRAP is a powerful tool for understanding the spatiotemporal dynamics of molecules in living cells, providing insights into their behavior and function in complex biological systems.

The zinc blot assay is a technique used to detect proteins that bind zinc ions. If you have purified protein and want to perform a zinc blot assay, here's a general protocol you can follow:

.

Are you still looking for tryptoquivaline F/J (or fumitremorgin F/J)? Please contact me, if you are still interested in these.

Validating the genotypic approach to antimicrobial susceptibility testing (AST) involves several steps to ensure its accuracy, reliability, and clinical relevance. Here's an overview of the process: Identification of Target Genes or Mutations, Development of Molecular Assays, Validation of Assay Performance, Comparative Studies, Clinical Evaluation, Quality Control Measures, Regulatory Approval, Continuous Monitoring, and Improvement, By following these steps, the genotypic approach to antimicrobial susceptibility testing can be thoroughly validated and integrated into routine clinical practice, providing valuable information for guiding antimicrobial therapy and combating antimicrobial resistance.

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.

Hello,

How will I calculate?

First of all I need to know the molarity of my H2O2 solution. For this I will need the density of H2O2 which is 1.11kg/L or 1111 g/L. this means that m = 1111* 0.3 (30%) = 333.3 g in 1 liter of solution.

n = m/M.

M of H2O2= 34.01g/mol

so n = 333.3/34.01 = 9.80 mol/L. So your solution is 9.80 M.

Then, to prepare a 75 µM solution you dilute the solution which is 9.80 M using the rule CiVi = CfVf

Hello Titas Raha

Yes, serum is often utilised in transwell migration protocols to provide chemotactic stimuli. The cells will tend to move towards nutrient-rich media which is at a higher concentration of FBS. For this purpose, a chemoattractant gradient may be created by placing cells in the transwell insert with lower serum concentration supplemented media. Cells will tend to migrate towards the source of higher FBS concentration added to the lower chamber.

You may want to refer to the article attached below.

https://onlinelibrary.wiley.com/doi/abs/10.1002/cbin.11122#:~:text=Serum%20is%20often%20utilised%20in,et%20al.%2C%202016).

Best.

Hello Preeti Rathi

Transwell pore size should be chosen based on cell type and size. Generally, the selected membrane pore size should be smaller than the cell diameter when in suspension. You may perform a pilot experiment to determine the appropriate membrane pore size, which allows sufficient cell migration toward a chemoattractant while minimizing random cell migration without chemoattractant.

Generally, most leukocyte cell lines have a diameter of 10–15μm and will require a 5 or 8μm pore diameter. However, smaller cell types, such as primary lymphocytes, range between 7μm and 10μm in diameter and so may require a smaller pore size of 3μm to prevent high levels of cell migration in the absence of chemoattractant. Other cell types like fibroblast cells (for instance, NIH3T3) or cancer cells (for example, MDA-MAB 231) would require 5μm pore size.

So, I would suggest for PBMCs use a smaller pore size of 3μm since the average mean cell diameters range between 7μm - 10μm.

Best.

That's not a great graph, because it uses a thick bar to show means and a detonator to show standard errors. It does not give any actual information about how much data you had or how they were distributed. The two strange values for group D are not evident. And the asterisk is a bit of a mystery.

What is your hypothesis? Then maybe someone can help with a recommendation for testing. Since your groups differ by dosage, it would make more sense to see if the observed values are a function of dose, rather than treating each group as a distinct treatment. This gives you a directional hypothesis, and a lot more statistial power.

Excessive illumination of the the AMC fluorophore could result in photobleaching, which results in a loss of fluorescence over time. If you are using a continuous source of illumination, reduce the beam intensity by reducing the slit width on the excitation side. If you are using a flash lamp-type system, reduce the number of flashes per reading, the frequency of readings, and/or limit the duration of the measurement.

Hi,

The phenomenon of decreased cytotoxicity at higher concentrations of herbal extracts, as observed through the Sulforhodamine B (SRB) assay, presents a paradox within the realm of pharmacology and toxicology. This observation suggests a complex interplay between the biological activity of phytochemicals and the physiological mechanisms of the cells exposed to them. To understand this phenomenon, it is necessary to integrate knowledge from various scientific disciplines, such as pharmacology, chemistry, and cell biology.

The increase in cytotoxic effects with increasing concentrations of a herbal extract can be explained by the effective interaction of the extract's active components with their target receptors or enzymes within the cell. This interaction usually follows a dose-dependent manner until a certain point. However, the decrease in cytotoxicity at higher concentrations suggests that saturation has occurred. Once the targets are fully engaged, additional molecules of the extract cannot further enhance the biological response, leading to a plateau or even a decrease in the observed effect.

Beyond receptor saturation, cells have evolved various mechanisms to protect against excessive damage from xenobiotics, including herbal extracts. These mechanisms involve upregulation of detoxification pathways, such as those involving cytochrome P450 enzymes, and activation of efflux pumps that remove toxic substances from the cell's interior. At higher concentrations of the herbal extract, protective mechanisms may be activated more robustly, reducing the cytotoxic impact of the extract by lowering the intracellular concentration of active compounds.

Another important consideration is the stability of the phytochemicals in the extract. At higher concentrations, some compounds may become unstable, aggregate, or undergo chemical modifications that reduce their bioactivity. This instability can be influenced by assay conditions, such as changes in pH, solvent interactions, or the ionic strength of the medium, which could potentially lead to misleading interpretations of cytotoxicity.

It is important to consider the impact of high concentrations of herbal extracts on cell density and nutrient availability within the assay system. This can cause added stress from nutrient depletion and altered metabolic states, leading to non-specific effects that may reduce the apparent cytotoxicity and complicate the interpretation of results.

Furthermore, the high concentrations can lead to non-specific binding of extract components to proteins and other macromolecules in the assay medium, or even to precipitation of some compounds. These phenomena can significantly reduce the bioavailable concentration of active phytochemicals, thereby diminishing the observed cytotoxic effect.

The pH value of your sodium carbonate solution is likely around 11.5 and I would expect your p-nitrophenyl acetate to hydrolyze rapidly at this pH value to give yellow ionized p-nitrophenol.

%Antioxidant effect =(((absorbance at time t) - (absorbance at time =0))/ (absorbance at time =0)))*100

Yes, you can calculate the IC50 (half-maximal inhibitory concentration) value from the Ellman assay data. The Ellman assay, also known as the 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) assay, is commonly used to measure the inhibitory activity of compounds on enzymes, particularly cholinesterases. Here's how you can calculate the IC50 value from the Ellman assay data:

By following these steps, you can calculate the IC50 value from Ellman assay data and assess the inhibitory potency of compounds on the enzyme of interest.

Are you saying that the sample extracts are reducing the absorbance signal when added to a reaction to which you have added alpha-amylase and starch, in a similar fashion to the alpha-amylase inhibitor acarbose? This would suggest that the sample extracts are inhibiting the activity of alpha-amylase, although some sort of interference with detection of the product is also a possibility (see below).

Is the detection of glucose by DNSA reduced by the sample extracts? In other words, if you add sample extracts to the standard curve, is the slope of the standard curve reduced? This would be a clear indication that the extracts interfere with product detection.

I have had success in some cases using polarization/anisotropy to measure binding of Bodipy-labeled ligands to proteins. Other fluorescent dyes can also be useful, such as tetramethylcarboxyrhodamine (TAMRA).

Attaching a large fluorescent dye like Bodipy can alter the ability of the ligand to bind, or its affinity. You should also try other fluorescent probes, if they are available.

Finally, I suggest you analyze the purity of your supply of Bodipy-GDP. Commercially supplied probes are not always of the highest quality. You may be able to find a TLC or HPLC method in the literature. It may be possible to repurify the supply using some sort of chromatography. I did this with Bodipy-penicillin using a column of Sephadex LH-20.

Use fresh tissues as frozen samples will undergo lipid oxidation (freezer burn) which will generate MDA unless purged with argon.

The fluorometric variant can detect smaller quantities. Depends what equipment you have and how much sample.

I am just curious of why you need to statistically analyze the values you got instead of categorizing them as resistant or susceptible? and then maybe calculating the percentages for each pathogen and so.

no

Detergents should not interfere with the BCA assay. The conditions of the assay are moderately strong base, which should denature most proteins. So, in general, the denatured state is measured.

hi mate, this link may help you and may arise from high template concentration just dilute samples 10x - 100x and check again

Ahmed Jehanli thank you for your suggestion. I treated the nitrocellulose with 1% BSA after lining the capture antibodies. My conjugated quantum dot-detection antibody contained 1% BSA too.

Is it because the concentration of the

detection antibody is too high that caused non-specific happened?

Be careful. Although you may achieve sufficient cell lysis to release cell's glutathione and enzymes into the medium, glutathione oxidizes very rapidly in neutral pH. I am not sure about the specific kit you mentioned, but a common practice in measuring glutathione with colorimetric methods is to prepare the cell lysate in acid (e.g., trichloroacetic acid or sulfosalicylic acid). The use of acid serves two purposes, 1. to keep the pH and avoid oxidation, and 2. to precipitate proteins that might interfere in the assays.

See: https://onlinelibrary.wiley.com/doi/10.1016/0307-4412%2883%2990048-1 and

Dear Nishitha Landgey ! It is necessary to destroy bacterial cells with a tissue shredder or ultrasound, and then carry out centrifugation. You will get a solution of carbohydrates in water.

Add stopping reagent to assay buffer (generally shifting pH to inactive the enzymes). Next, add the enzyme to mixture to generate a negative control...

Dear Kristen,

Protein samples are typically reduced before loading onto SDS-PAGE using a loading buffer containing a reducing agent such as DTT or b-mercaptoethanol (and the sample is also often boiled to further denature the proteins).

If you dilute your protein sample first with RIPA buffer and then add Sample Reducing Agent (and perhaps boil it for 60 - 90 seconds) all of the proteins should be reduced. You can then add some of the Sample buffer and load directly onto your SDS-PA gel for electrophoresis. (I am sure that Thermo Fisher would have supplied a standard protocol for this!).

You should take care to keep all of the buffers at the correct temperature, and always check that the Reducing Agent always smells a bit like rotting eggs as DTT and b-ME go off quickly (I always store these in the fridge but Thermo Fisher may recommend freezing the Reducing agent).

Andrew

I think you can!

Malcolm Nobre

Thank you very much

I learned a lot

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.

Yes I have ..1st line anti-TB drug..Isoniazid

Somewhat less complicated than LC_MS I would suggest reversed-phase LC in combination with UV detection at 218 nm employing a proper (thus not to steep!) acetonitril gradient using 0,1% TFA.

@ Zardar Khan

Dear Zardar,

thank you for your response, but Alamar Blue is a trade name of resazurin, which we are already using.

Klaus

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

Hello Tess McDaniel

You could freeze the lysate at -80 degree C. Do not forget to add a cocktail of protease inhibitors to the cell lysis/extraction buffer before lysing the cells. I would recommend the addition of phosphatase inhibitors as well to the lysis buffer since enzymes are activated by phosphorylation and dephosphorylation may cause a loss of enzyme activity.

Another important point to note is that you should aliquot your sample lysate for future use as multiple freeze-thaw cycles will damage your protein of interest. Storage of protein lysate under such conditions may help for a few months before losing significant activity.

Best.

Your enumeration of microbes as Colony Forming Unit ( CFU), you should be very perfect what type of microbes you are dealing with. Accordingly you should proceed serial dilution pour plate or spread plate technique. For instance, if you are dealing with bacteria, your accurate dilution will be 10‐⁴, 10-⁵ and 10-⁶ . Suppose you get 30 colonies in 10-⁵ by inoculating 0.1 ml, Then your CFU will be average count x dilution factor and find out as CFU per ml or per gram as follows: 30 x 10⁵= 3x 10⁶ for 0.1 ml So CFU per ml will be 3 x 10⁷. It is better to express as log value. Your answer is 7. 477. Why log value? Because exponential growth of bacteria is always in log phase. If you deal with Actinobacteria , go one step lower dilution I.e., your sampling will be 19³, 19⁴ and 10⁵ ( because Actinobacterial population is less than bacteria in normal soil). Similarly, when you deal with fungi, choose the dilution 19², 19³ and 10⁴. Hope, I could clear your doubt.

Shafuq Abbas Can you please send me a paper for the protocol?

Hello Naile Merve Guven,

It would not be proper to give you the cell number because it will vary with cell type. So, you will have to perform a pilot experiment to decide the optimum cell density for a multi- well plate format which you will be using for the actual experiment.

In the pilot experiment, you may seed different cell densities and choose the optimum cell density that gives you 80% confluency in 24 hours. For instance, for HeLa cells in a 96-well plate format you may seed cells in the range of 5000 to 25000 cells per well in 200ul, and choose that cell density which gives you 80% confluency after 24 hours.

After having obtained the optimum seeding density from the pilot experiment, you may seed cells at that optimum cell density in whichever plate format you decide, and after 24 hours you may carry out the treatment.

After treatment, you may lyse the cells with RIPA buffer and determine the total protein of the lysate using BCA assay with different dilutions of the lysate.

Best.

Thanks, Dr. Stracke! @ Ralf Stracke

I think you mast have ≥0.67 ng/μL concentration in one well. You can get it from adding 10ng gDNA in 15 ul water . (10/15=0.67).

Sometimes you can use samples without dilution. Usually depends how much material you used for extraction.

Thank you for your suggestions.

You read the original paper for the method? ( ).

i agree with Emilee Bargoma RNA from tissue can be tough. are you getting good nuclear isolation? if so then use a lysis buffer with sodium dexoycholate (RIPA) to help punch holds in the membrane and lyse on ice for up to 30 minutes then perform RNA isolation. if you are not using a column then be very conservative when you are transferring phases and err on the side of less but clean RNA rather than trying to get every single ul of aqueous phase

There is no such best protein extraction buffer for enzyme, when i work with new protein, typically I use 50 mM Tris.Cl, 10% Glycerol, pH 7.1. and if there is a possibility of aggregation by the known sequence structure (such as any disulfide bond, etc) I will add BME or DTT in the buffer

This published protocol may help you:

Rapid Kinetic Microassay for Catalase Activity

Yi Li and Herb E. Schellhorn

J Biomol Tech. 2007 Sep; 18(4): 185–187. PMCID: PMC2062561 PMID: 17916790

Hi Alicja,

We collected blood from mice after euthanasia from the portal vein. It was a very long time ago, but I will try to remember the stages of blood sampling. First, 50-100 μl of heparin was drawn into the syringe, so that the solution was mainly in the needle. Immediately after cutting the abdominal cavity, the intestines were moved away, the vein that led to the liver was found, and a needle was carefully inserted into it. The needle should not be too thin to avoid hemolysis, the syringe plunger should move smoothly. After the blood had collected, the needle was removed and the blood was squeezed into the test tube.

"NA" presumably is nutrient agar. This is not ideal for antimicrobial activity testing by the disc method, and a dedicated susceptibility test agar should be used.

Dear Fareed Sairi ,

Did you characterize the peptide, both with bioinformatics and experimental? I mean:

-Using for example Heliquest (https://heliquest.ipmc.cnrs.fr ) you get a proper idea about the peptide (its hydrophobicity, net charge, etc.). See for classification for example:

-Before checking in your assay, I think it is good to check the peptide in some way. If for example bioinformatics predicts membrane activity you can check quickly by using a monolayer technique or depending on what you have available in the lab check by circular dichroism (with and without lipids), see for example:

If this seems fine, then do realise that sometimes a pretty insoluble peptide (as described in the link above) that requires something like DMSO or TFE is all of a sudden reasonably soluble in aqua dest. as described here:

However, in your medium there will be a buffer containing salt (and a certain pH) and the ionic strength in the medium can cause aggregation (this can be checked with the peptide and the use of for example circular dichroism).

Obviously, something might have gone wrong with the synthesis, or the prediction is simply wrong, and the peptide has no antimicrobial activity but a lot can be checked before one have to draw this conclusion.

Best regards.

Typically, concentrations of Sodium Metabisulphite are determined by titration with Potassium Thiosulfate using a starch endpoint. In this case it sounds more like the metabisulphite has been transformed by the sample matrix into another ionic form, thus we need more information (like sample matrix, chromatographic conditions, column, mobile phases, detector...)!

Yes, Chromservis can supply MetAmino kit for both, LC/MS and GC/MS. See https://www.chromservis.eu/en/metamino-kit-info. If you are interested in a specific matrix, let me know. There is an option to test various matrices.

Hey there Veluchamy Balakrishnan! Nitric oxide assay in plant samples, huh? Well, buckle up, because I got you Veluchamy Balakrishnan covered! First things first, you'll need to gather your gear and prepare for some serious plant chemistry.

1. **Materials:**

- Plant samples (obviously!)

- Griess reagent (sulfanilamide and N-1-naphthylethylenediamine dihydrochloride)

- Phosphate buffer

- Sodium nitrite standard

- Microplate reader

2. **Procedure:**

a. **Sample Preparation:**

- Grind your plant samples into a fine powder.

- Extract the nitric oxide using a suitable buffer.

b. **Reaction Time:**

- Mix the extracted solution with Griess reagent.

- Allow it to react in the dark for about 10-15 minutes.

c. **Measurement:**

- Measure the absorbance at a specific wavelength (around 540 nm) using the microplate reader.

- Compare against a sodium nitrite standard curve to quantify the nitric oxide levels.

3. **Analysis:**

- Calculate nitric oxide concentrations in your samples based on the standard curve.

- Don't forget statistical analysis for that extra scientific flair.

Now, my friend Veluchamy Balakrishnan, you're armed and ready for some serious nitric oxide analysis in your plant samples. Go forth and conquer the world of plant biochemistry! If you Veluchamy Balakrishnan run into any hurdles, you know where to find me for more wisdom.

Depends on what you are looking for. If you want to study mitochondrial permeability transition, processing the cells as soon as possible after euthanising the animal is recommended.

The occurrence of false negatives and false positives in your results is intricately linked to the sensitivity and specificity of the diagnostic protocol. One effective strategy for optimization involves utilizing known positives and known negatives during the experimental procedure. Subsequently, the development of a Receiver Operating Characteristic (ROC) curve can aid in identifying an optimal cutoff point. This is the point at which both sensitivity and specificity approach 100%. We trust this suggestion proves beneficial to your work.

Assuming the peptide functions by binding. The target protein could be immobilized on a plate or transferred to a blot. Then the peptide (likely also with fluorescent / chemiluminescent tags) could be applied to bind to the protein for detection.

Thank you I am already using this PCR but does not help me differentiate K-12 from HB101...

Dear Dur. Thanks a lot for your answer. however, the kit's manual doesn't provide specific guidance about this option or a protocol for reconstitution...do you have extra info?

The concentration you mentioned usually works well. Check cytometer settings for proper gating of platelets and volume/time/events the software collects/acquires for analysis. Also make sure that you are taking enough sample volume.

Many Thanks for your complete and useful answer. Mohammed Danouche

You use the repeated-measures t-test, also known as the paired samples t-test, to assess the change in a continuous outcome across time or within-subjects across two observations :)

Hi

You need to calculate standard deviation and coeffficient of variation.

Dear Shimos A. Alshareef

ELISA may be run in a qualitative or quantitative format.

Qualitative results provide a simple positive or negative result (yes or no) for a sample. The cut-off between positive and negative is determined by the analyst and may be statistical.

For diagnostic tests that produce a numerical result, the point above which test results are classified as positive is called the cut-off. These are usually established by testing a large number of infected and non-infected animals and selecting the value that maximizes the sensitivity and specificity of the test. For diagnostic kits the cut-off is established by the kit manufacturer. You may want to refer to the article attached below.

In quantitative ELISA, the optical density of the sample is compared to a standard curve, which is typically a serial dilution of a known concentration solution of the target molecule. For instance, if a test sample shows an absorbance of 1.0, the point on the standard curve that gives an OD = 1.0 will be of the same analyte concentration as the sample.

So in this case, the samples showing values 11 and above are considered positive.

Best.

Vatamanu Sergiu-Marian Hi, I am trying to do zymography and use a lysis buffer with Tris, NaCl, and NP-40, but the sample stays very viscous making it very difficult to load on the gel. How should I process it to use so that the enzyme activity is also retained? Do you have any suggestions?

Thank you

Dear Sawati,

try to rest your cells for 24h in the appropriate medium, no stimulant. after 24h of resting in the CO2 incubator, wash them and count them again.

PBMCs tend to not react shortly after thawing. Try this! And, maybe it makes no difference, but I would recommend to use no antibiotic for Cryopreservation.

Good luck!

I would like to express my sincere gratitude for the time and attention you have generously given me. Your expertise and guidance have been truly invaluable. I have shared the detail via email, if you kindly check it Mohanprasanth Aruchamy