Enzyme Activity - Science topic

Hi, Ive recently conducted a thermal shift assay on my protein sample. My protein is in 10mM phosphate buffer with 0.1 % Triton X-100. for the record i use the default setting on Biorad qpcr following the manual except that i use the non label method (intrinsic fluorescence of Trp)

After the run, my sample yield a result like the attached picture. The bottom black and magenta line belongs to my buffer control and the rest are the sample and replicates.

I wonder if my protein aggregated and denatured considering its an old sample with few freeze and thaw cycle. thats why the initial cfu reading is higher than at the end with no peak at all.

Why does my thermal shift assay produce melt curve like this?

I had done the abs vs conc std curve. But what is the correct formula to be used to find the enzymatic activity?

Apart from this std curve, do I need to plot another graph abs vs time for my protein sample to cal delta abs/time ? Does it somehow related to find the enzymatic activity?

I am currently conducting research and came across the unit PROT/g. However, I am more familiar with expressing enzymatic activity in units (U) and would like to understand the meaning of PROT/g and when it should be used instead of U.

Thank you

Hello!

I want to quantify HMGCS2 enzymatic activity in a simple way and I wondered if someone knows of an in vitro enzymatic activity protocol using spectrophotometry or similar.

Thank you!

I have to quantify the enzymatic activities of SOD in plants using a method that employs hypoxanthine-xanthine oxidase to create O2. The colorimetric reaction was performed by combining alpha-naphthylamine and sulfanilic acid. I measured the absorbance at 550 nm and recorded the absorbance, including a blank without the plant extract to determine the maximum production of O2, as well as the absorbance of the plant extract.

Based on this information how can i calculate activities of the SOD?

Is there a method that I can measure alcohol dehydrogenase activity using NAD+ in enzyme extraction from plants?

Protein content in malt processing can have several effects, primarily related to its impact on the enzymatic activity during mashing and its influence on the final characteristics of the malt and the resulting beer.

I have purchased Acetylcholiesterase from Electrophus electricus (electric eel), C3389-2KU, following details are written on the enzyme vial.

Type-VI S lyophilized powder 200-1000 Units/mg protein, 374 units/mg solid, 610 Units/mg protein, 5.3 mg/solid.

i want to prepare 0.28 U/ml working solution, please help me with the preparation of stock solutions and the calculations.

I am doing a carbonic anhydrase activity assay using p-nitrophenyl acetate.

reaction conditions: 37 °C for 10 minutes

After 10 minutes of reactions, I noticed a yellow color in all the reaction tubes except the blank, which remained colorless. To stop the enzyme reaction, I added 800 ul of 2M Na2CO3 to all the tubes. Surprisingly, even the blank changed color to yellow. As a next step, I examined the absorbance at 400 nm. the results obtained turned out to be inaccurate.

Hi all.

I ran the enzyme activity (pure Tyrosinase after purification using recombinant e coli) using L-Dopa with this condition

50 mM phosphate buffer ph 6,

1mM L-Dopa

25 C temperature

0.5 mM sls for enzyme activation

Result was positive with appearance of pink solution.

I did kinetic parameter with specific activity, which all show positive result.

However, when i started to do different temperature activity, now there was no colour change ? So, i repeated the same activity test also no colour change.

So, has anyone experience the same ? Attached is the activation test that showed pink solution.

In biochemistry, most work is done at room temperature. Yet, basic thermodynamics tells us that affinities often change with temperature, in positive or negative directions depending on the entropy/enthalpy contributions. Thermal transitions can occur which both quantitatively and qualitatively change the behavior of the molecules of study. In enzymatic reactions rate-limited by diffusion-mediated product release, the increased rate of diffusion could increase the rate of product release above the rate of the chemical step, such that the chemical step becomes rate-limiting. If one discovers a compound that potently inhibits this enzyme at 25C, it may have reduced effect at 37C, or none at all. Likewise, if an enzyme is predominantly dimerized at 25C based mostly on enthalpic contributions, this dimer may not even exist at 37C. Screening compounds against the dimer may be of little relevance to the situation in vivo. The converse could happen if dimerization is entropically driven. Temperature-dependent changes in solution properties can also obscure the relevance of 25C results to 37C, such as viscosity.

I welcome everyone's two cents.

Is it correct to filter the extracts we obtain with Whatman filter paper and use them directly in experiments to investigate the bioactivity of phytochemical substances? Or is it correct to first centrifuge the extracts we obtain, then filter the supernatant and use them in experiments?

attempted the crosslinking method using glutaraldehyde, which resulted in pellet formation that is challenging to dissolve, possibly due to excessive crosslinking. I'm now seeking an alternative approach.

Hi everyone,

sorry if this is a simple question, or if I get anything wrong here, but I'm very outside of my comfort area when it comes to enzyme activity-related calculations.

So, I need to know the concentration of enzymes that a paper used for their stock solution of Catalase.

Here is a quote from the methods: "Enzyme activities of stock solutions were 3 mM/s for GOX and 998 s-1 for CAT. To obtain a defined, stable oxygen concentration of 2% on cell surface stock solutions were diluted by 1:10,000 for GOX and 1:1,000 for CAT."

For the GOX, I think I can manage to calculate the stock, but the catalase is the issue

So, the Kcat = 998 s-1

The Vmax = 1uM/ min = 0.0166uM/ s

For for the calculation: Kcat = Vmax / E[t] , is it as simple as rearranging it to E[t] = Vmax / Kcat?

That Vmax figure is from the data sheet of the catalase, with 1 unit being equal to 1uM H2O2 processed per minute (not 100% sure this is what is meant by Vmax), hence me dividing by 60 to get the uM per second.

I also know that 1mg of Catalase = 20,000U

The issue is that I don't know how to put this all together. I am currently trying to get more familiar with enzyme kinetics, but this is taking some time. I would very much appreciate if anyone could offer some advice to help speed things up so I can start with my experiments.

If I am missing some information here please let me know.

Best regards,

Ciarán

Hello everyone it will be of great help to me as I am working on my dissertation, I have enzyme activity in mg/ml for papain, just need to calculate it in percentage.

Thank you.

I try to measure the enzymatic activity of catalase in a symbiotic Cnidarian,

I homogenized my tissue in 0.1 M phosphate buffer by two methods

1- using fast Prep with beads

2- sonication for at amplitude 20% and even more for longer time period.

then I noticed microscopically that both of theses do not results in significant algal cell lysis

My enzymes are SOD and Catalase,

What is your suggestion. Please

given that the lysis buffer should not affect the enzymatic assays.

I am a bit confused about the matter. The majority of papers and explanations I have come across indicate that the inhibited line is generally positioned above the uninhibited line, but I have observed it to be the opposite. will it still be considered as lineweaver burk plot?

I have some problems with doing the calculations for protease enzyme activity after using this protocol.

Universal Protease Activity Assay using Casein as a Substrate (sigmaaldrich.com)

Is anyone familiar with this protocol and can offer some assistance on the calculations? Any assistance would be appreciated. If i have an idea of how it's done, I will be able to complete the rest of my calculations. The explanation for doing the calculations is a bit confusing.

Thank you in advance.

Also, to those who answered my last post thank you. I was able to grasp both Amylase and Lipase enzyme activity calculations.

Recently I am stuggling to improve the kinetics of an artifical enzyme. I expressed this enzyme in E.coli and then test it's kinetics properties.

I noticed that if I pick single conies for testing, there will have a variation in both reaction rate and maxium reaction. indicating in fig.1 (the y axis represent the product that have been formed and the x axis represent the time in seconds.) 4 different conies have been picked and they all contain the same plasmid that transfection at the same time and same procedures. However there is huge differeces in the reaction rate and maxium reaction.

Then I wonder if it's due to the different conies would fold the protein differently, so I did another test by add multiple conies (actually all conies on one dish) into my culture medium. And then I test this mixed enzyme with different substrate concentration to test the affinity and kinetics at the same time. fig.2 (different color represent different concentration; the dash line represent a Imaginary limitation)

The problem that makes me wonder is that: what might be the reason for this reaction have a rate limitation?

I have few hypothesis about this phenomeon:

1. based on the Imaginary rate limitation; there might have steric effects preventing the binding of the substrate. (but I don't have see enough enzymatic reaction curve that have steric effects)

2. based on the varation between conies; this artifical enzyme might have many different ways of folding (I mean this enzyme would have many different prefered structures in different bacteria cells). maybe bactria from the same coniey would prefere similar stucture? and some stucture have better enzymatic performance, others do not.

I am really appreaciarte your reading and would be very happy to receive any response.

For the enzymatic activity assay, I have to use 4-Hydroxybenzoic acid. I have kept it in the refrigerator. In the first experiment, I saw one peak but, after 3 weeks I was 2 peaks during UV spect.

Do you have any idea or guess?

Thank you

I am currently doing an experiment and working on amylase, lipase and protease enzyme activity. I have followed various protocols and have done experiments. I am looking for assistance in doing the calculations for the enzyme activity. Anyone with said expertise, I would appreciate your assitance.

I have collagenase from sigma product code c9891 100mg,Type IA, 0.5-5.0 FALGPA units/mg solid, ≥125 CDU/mg solid, For general use

I do not understant from the product data sheet how many units are per mg (U/mg)....

How can I make a 30U/mL solution? How much buffer should I add to what quantity of powder to obtain 100mL at 30U/mL solution?

I would appreciate your experience. Thank you!

I basically have normalized fluorescence data generated from a series of protein dilutions. I need to calculate K, K should be the same in all dilutions, thus the need for a global fitting approach. I have been using XLfit, but would like to move into a script format. Thanks in advance! Thus far, I found the package renz, but to my understanding, it does not accomodate for global fitting. Thanks in advance!

Hi everyone,

I am measuring the activity of some mitochondrial enzymes, by measuring the oxidation/reduction of relevant substrate using spectrophotometer. However, after analysis, my enzymatic activity (nmol/min/mg) becomes a negative value (not always, but most of times).

Is this because my analytes are being used (thus decreased) so their absorbances decrease?

Another question; if the extinction cofficient of my analyte in 340 nm is 6.22 mM-1.cm-1, and the length of cuvette is 0.6 cm, and the concentration of my analyte is 0.1 mM, how should I calculate my enzymatic activity in nmol/min/mg?

Big thanks in advance

I have the commercial cellulase product (100mg) with 13000 U/g. I need to know how to convert this U/g units into FPU unit for saccharification process with lignocellulosic biomass. Thanks in advance.

I know that metal ions are present in enzymes, and that this affects their stability and function.

However, when producing or engineering enzymes, I wonder if metal ions have a negative effect.

Metal ions are interacting with cysteine and histidine in proteins such as zinc finger and ring domain.

Are there cases where cysteine and histidine are replaced with other amino acids to remove metal ions and maintain the structure?

I'm working with simulated digestive fluids.

Due to the low concentration of pepsin that I need for my work, it is difficult for me to weigh very small amounts of the reagent each time I need to use it (in fresh). That is why I decided to prepare a stock using 50 mg, from which I can take aliquots whenever I need it. But I don't know if it affects the fact of defrosting it continuously every time I need to use it.

I diluted the powder of Pepsin in HCl 0.01 N. I didn't find info about the appropriate storage temperature.

Also, does anybody know if its correct to store the solution in HCl 0.01N?

I emphasize on this because a found a protocol that says: "A stock solution of Pepsin is dissolved in 10 mM Tris buffer, 150 mM NaCl, pH 6.5. The stock solution has to be stored on ice or refrigerated at 4°C. Just before the assay, the concentrations of pepsin in 10 mM HCl has to be prepared".

From this, I understood 2 points:

A) The pH is adjusted to 6.5 to limit proteolytic activity

2) Storage on ice limits proteolytic degradation.

But, as I mentioned before, weighing small amounts increases the systematic error. That is why it is more practical for me to have a stock and keep it in storage.

In B.subtilis spore surface display cot partner with native promoter is fused in frame with a passenger protein via a linker like GGGGS. The assumption here is that a fusion mRNA is formed, and translation starts from the cot partner START codon and ends at the stop CODON of the passenger protein. The linker permits the independent folding of the two proteins. My question is will omitting the N terminal START codon of the passenger protein (enzymes in my case) affect its stability and activity? I'm having multiple enzymes fail with multiple cot partners like cotB,CotC and CotG. I have not included the START codon in the passenger protein in my constructs. I have been checking the literature and so far in every study they have included the start codon of the passenger protein. Any thoughts about its impact?

I have to conduct enzyme assay of around 200 samples to check their kinetic rate.

Traditionally, for small number of samples we carry out cuvette based spectrophotometry assay. But for such large number of samples carrying out individual spectro analysis will be time consuming and chances of error. It will be helpful if any alternative and parellel analysis technique/protocol suggested.

From the practical experience I have observed, when I have added extra amount of tissue homogenate (more than recommended) to study any enzyme activity (for low expressed enzyme assay), the initial absorbance starts with very high value (some times nearer to 1) and never gets satisfactory result. Moreover, excessive amount of tissue homogenate makes the rection mixture colored (similar to tissue homogenate color).

So, what should the balanced amount of homogenate should I use for any enzyme activity assay?

How do the excessive amount of homogenate affect the reaction?

Thanks in advance.

Dear all,

I got results after performing an enzyme kinetics with 4 different substrate the kcat of B and D is respectively two times lower than A and C. However, I am seeing the higher Km of A i.e 174 nM as compared to B i.e 54 nM. Can anyone please suggest me how to rationalize this result ? The Kd value however, is always doubled for A and C when compared to B and D respectively. Below are the data attached for your reference. How could it be explained the research paper ?

Thank you

I am preparing gastrointestinal fluids to test yeast survivability in-vitro.

The protocol requires me to prepare two solutions, gastric juice and intestinal fluid, requiring me to add pepsin and pancreatin respectively after the autoclaving.

There are MCE, PTFE and PVDF filters available in the lab, and my concern is possible loss of enzymatic activity due to using unsuitable filter. Which one, or another, do you suggest for preparing such solutions?

Does the stability change with stock concentration?

Hello,

This might be a very ridiculous general question but here we go:

I am trying to find kinetic parameters for the metabolism of a drug by the enzyme CYP3A4. The problem is, all literature Vmax values are specific activities either in pmol/min/mg or pmol/min/pmol CYP3A4, but I need Vmax values to be in the form: mol/h or mol/(h*L) ( for a systems biology application that uses these units in rate laws)

I am not an expert on enzyme kinetics, so I am not sure how one makes the conversion to fit the Vmax data into a this format. Do we need the enzyme concentration (in g/L or in M), as well as the incubation volume?

Some insight would be highly appreciated.

Do we need to test the enzymatic activity of the supernatant and the wash too? and what is the differences between recovery and residual activity?

In a hypothetical scenario, researchers discovered a protein that is responsible for lactose intolerance in cats. The results of the multiple sequence alignment for these two proteins are as follows:

Different parts of the protein were cloned and expressed in vivo in a mammalian cell. The enzymatic activities for different parts of the protein are summarised in the table below.

a. Which part of the amino acid segment most likely contains the active site? [1 mark]

Your answer here.

b. Which construct will you generate to produce more than 85% enzymatic activity?

Justify you answer. [3 marks]

Explanation in bullet points (25-50 words).

c. Describe the protein engineering strategy/strategies and steps to generate this new construct. [3 marks]

I am working towards to design a study to asses the efficacy of different LRRK2 inhibitor compounds in neuron. I have been meaning to ask if there is any gold standard to check LRRK2 enzymatic activity.

Thank you

Hi

The question is: Determining hexokinase activity in rat brain where 10 µl of a 10% (100 g/L) brain homogenate (containing hexokinase) is mixed with 1990 µL of reaction medium (containing the substrate glucose and the coenzymes ATP and NADP+ and the auxiliary enzyme glucose-6-phosphate dehydrogenase). The reaction mixture is placed in a cuvette (light path = 1 cm) in a spectrophotometer at 37°C, where the hexokinase reaction can be seen as an increase in absorbance at 340 nm. The reaction proceeds at a constant rate during the measurement period, and during 10 min a total increase in the light absorption at 340 nm of 0.10 is recorded. The absorption coefficient of NADPH = 6300 x M-1 x cm-1

I need to find the enzyme activity of hexokinase in µmol/min/g. I don't understand how I need to interpret the first line (10 µL of a 10 % (100 g/L) brain homogenate) - and how should I use these numbers?

Thanks in advance!

I am working in laccase enzyme production by bacteria. In that i have a problem with quantitative estimation of laccase enzyme. I have confirmed the laccase production with plate assay using guaiacol. But i face the problem with quantitative method of laccase enzyme. So kindly suggest me some tips and methods for the laccase enzyme assay. Thanks in advance:)

I found the dns method to measure the amylase activity, but I cannot get the dns reagent. Therefore, could you suggest me an alternative method or alternative reagent to dns reagent?

Sometimes the enzyme may not be activated or may not function properly unless the it is co-expressed with other protein for post-translational modification or assembly into a functional catalytic complex.

Does a better docking score or stronger binding energy between a mutant enzyme with its substrate always translate into an improved enzymatic activity?

How should RMSD, RMSF, radius of gyration, solvent-accessible surface area (SASA), etc. from molecular dynamics simulation be used to guide enzyme engineering with the aim of improved product synthesis?

Hi, Iinserted gene of Reverse transcriptase into pPLc245 plazmid. If I'm right, this plazmid doesn't code cI857 represor. For expression od reverse transcriptase from this plasmid I used E. coli DH10B. RT was expressed after increase of cultivation temperature to 37 °C. Before this thermo induction I cultivated E. coli DH10B with pPLc245 at 28 °C. But at this temperature RT was not expressed. Why is this possible? Is E. coli DH10B coding cI represor or pPLc245 could contain gene for this represor?

Thank you for all responses.

Bohuš

Hi, every body

I am investigating the enzyme profile of a white-rot basidiomycete fungus. In the enzyme assays, we detect enzymatic activity by color change. However, my fungus produces pigments that do not precipitate with centrifugation, so it causes errors in the results. Does anyone have a solution? Thanks.

I would like to ask you, when I measured the enzyme activity of papain by the national standard method, but the sample tube and the blank tube became zero and negative values when subtracted from each other. The blank tube was added with TCA first, which means that it was caused by the casein itself and showed a light blue color.

How can I adjust the OD value of the diluted papain, which is also light blue, to 0? Is there a problem there?

There is no air bubbles in the measurement, and the instrument operation and personnel operation have been excluded.

Translated with www.DeepL.com/Translator (free version)

I am about to calculate alpha-amylase activity for some fungal samples. I followed the protocol of Sigma-Aldrich (https://www.sigmaaldrich.com/DE/en/technical-documents/protocol/protein-biology/enzyme-activity-assays/enzymatic-assay-of-a-amylase) and incubated my samples in 0.2 % starch solution for 3 minutes.

According to the protocol, the equation for the U/ml value is:

U/ml = (mg of maltose released * dilution factor) / (ml of enzyme applied)

I think I also have to consider the incubation time of 3 minutes, and divide my result from the equation by 3?

Thanks in advance for help.

I have been failing to express a very large trypanosome (160kDa) protein using various expression systems. The protein is a chimera of 3 enzyme activities and I can express truncated forms containing at least one of these enzyme activities. So, I was wondering if I can express and purify these enzyme 'domains' individually and somehow get them to associate, in order to perform enzymatic assays assessing their potentially interactive (regulatory) roles.

For instance can an inducible di-cre recombinase-type system be used? Ideally any recombination domains (or tags) that I may fuse to my recombinant construct would be as small as possible.

I have diluted my chrysin in DMSO, however when run an enzymatic assay in phosphate buffer pH 7.4 (50 mM) and pH 7.6(10mM) it formed precipitation? do you have any suggestion to stop it but still using the same buffer? Thank you

The Vigna radiata plants were subjected to Cd stress ranging from 50-200 ppm. Why does the enzymatic activity (SOD, APX, and CAT) increase under increasing metal stress despite the decrease in protein content?

Hi, I'd like to ask whether it is necessary to precipitate MMLV Reverse transcriptase before affinity chromatography purification (Äkta)? My colleague must do this step with his Taq DNA polymerase. He use (NH4)2SO4 or Na2SO4 + PEG. Without this precipitation is polymerase inactive.

Thank you for your responses.

Bohuš

How to know whether a particular protein is an auto cleaving protein or not (i.e. Self cleaving) ?

Also suppose a particular protein is degrading immediately so How to know whether this is due to Auto cleaving nature of the protein or due to some other proteins (i.e proteases) in the supernatant.

Hi, has anybody experience with addition of ammonium ferric citrate to expression media? I decided to ide this because of its perfect solubility in water.

Thanks for your responses!

Bohus

Hi everyone, I have been trying to calculate the K_m value of a recombinant HCV NS3/4A protease using a FRET substrate, the cleavage of which can be detected at 500 nm using excitation wavelength of 355 nm. I have a RFU vs. time graph of different concentration of the substrate (attached). Next, I am calculating the initial rate which is the slope of the linear initial part of the progress curve, with the ultimate goal to plot the 1/[S] vs. 1/[V] graph and calculate the K_m from the Lineweaver-Burk equation. However, if I calculate the slope from this graph for the time range between 0-10 minutes which is the initial linear part, the intercept for the 1/[S] vs. 1/[V] graph is negative but K_m value can not be negative. I was wondering if anyone can guide me on how to calculate the initial velocity correctly from the graph I have? Thank you so much!

Can I say for a given same enzyme, more number of substrate A is changing into the product than substrate B per second ? Please make this this this clear to me. I know Kcat/Km characterized high efficiency. However, I am getting into this situation somehow. Is it possible ? How I would justify or rationalize this in the paper for publication ?

I am a student from a relatively developing country and the commercial enzyme is expensive for us. My graduation thesis is desperately in need of an available standard curve for my AchE assay so that I can calculate my results using it. Please is there anyone who currently doing the AchE assay who can share it with me or where can I find it, are there papers or textbooks that I can find it in?

Your help would be very much appreciated.

Thank you

I can only start with raw feather from chicken/duck, but not feather powder.

I'm trying to run an assay for Caspase 3/7 activity to visualize apoptotic cells in flash frozen, unfixed mouse skin sections. The assay involves a non-fluorescent reagent that is processed into green fluorescence by endogenous, active Caspase 3 or 7 in apoptotic cells.

After thawing the slides for 3 minutes and washing once in PBS to remove OCT, I added the reagent to the unfixed tissue (I've tried 1 hour at room temp, or 30 mins at 37C). After the incubation, I post-fixed the sections with 4% paraformaldehyde, added DAPI mounting medium then visualized. 100% of the cells had green fluorescence, and the nuclei appeared enlarged so it looks like the cells are bursting or autolysing during the incubation. I'm wondering if I should fix the tissue immediately after thawing to stop the damage to the cells.

What would be the best method for post-fixing flash frozen mouse skin so that endogenous enzymatic activity is maintained? Does anyone have experience specifically with using the CellEvent Caspase 3/7 assay from Invitrogen in frozen tissue sections?

PS. I have also tried a TUNEL assay which generated no signal.

Most people say that imidazole doesnt affect the majority of downstream applications for purified proteins but it is a chelator, so you would think that it might chelate the Mg2+ in solution and inhibit Mg2+ dependent reactions. Anyone seen any papers that discuss what concentration Imidazole will chelate magnesium or manganese ions in solution?

Hey guys,

I'm using (for me new cells) E. coli Arctic express for protein expression (reverse transcriptase). I tried expression at 20°C and 10°C. At 20°C solubility of my proteine was 50% and at 10°C it was about 57%. I'd like to know some your experiences and tips how to use these cells (the best media for them, optimal temperature for night culture or growth up to induction and after induction, how to eliminate chaperonins after expression, how long should lasts expression, some supplements to media, concentration of antibiotics in night culture, concentration of IPTG ... )?

Thank you for all advices!

Bohus

Hello everyone,

I'm working in my doctoral thesis in the purification of a lipolytic enzyme from an halophile archea. Currently, I'm trying to purificate the recombinant enzyme using Haloferax volcanii as an expression system. The problem is that suddenly when filtering my crude extract through a 0.45 um nitrocellulose membrane I lose 90 % of the enzymatic activity, when that did not happen before and at most I lost 20 % of the activity.

Has the same thing happened to someone else? Or do you have any ideas that could help me? Thanks!

We are used the protocol for urease extration adaptated (to C. neoformans) from Amin et al.,2013:

Briefly, the broth cultures were subjected to centrifugation (5,000 × g, 4 °C) and the recovered mass was washed twice using phosphate-buffered saline (pH 7.4) and then stored at -80 °C. Subsequently, was thawed to ambient (room) temperature, followed by mixing with 3 mL of distilled water and protease inhibitors (TLCK, Tosyl-L-lysyl-chloromethane hydrochloride) and sonication for 60 s. After centrifugation (15,000 × g, 4 °C), the supernatant was desalted by eluting through SephadexG-25 column. The resultant crude urease solution was mixed with an equal volume of glycerol and then preserved under refrigerator (4 °C) for further uses.

Okay, we ran the suggested protocol, but after 2 days, enzyme activity was no longer observed.

Then, we tried to exclude the TLCK from the protocol, again the activity was observed on the day of extraction, but after it lost the activity.

We also tried to activate urease with sodium bicarbonate and Ni solution, without success.

In another procedure, we repeated the protocol and lyophilized the solution resulting in a white solid. So we tested it on this day, we prepared a urease solution (10 mg / mL) and the activity was good, but after a few days, again the activity was lost.

We always work with refrigeration, and we are careful to keep the solution on ice when we handle it.

Please any suggestions?

Hi, I did heterologous expression of reverse transcriptase in different strains of E. coli at 3 temperatures, 37 °C, 28 °C and 20 °C. As I expected, BL21 provided the best solubility at 20 °C and the worst at 37 °C. On the other hand strain MC4100 had the best solubility at 37°C and the worst at 20 °C. Has anybody similar experience with E. coli MC4100?

Thanks for responses!

As I am characterizing an 3' exonuclease and calculated the apparent Km and Kcat for two different substrates. What I observed is little bizarre. A substrate (S1) which looks like (qualitatively) degraded slowly by the enzyme has Km lower (~60 nM) and lower Kcat (5.1 sec-1) while other substrate (S2) apparently degraded faster, has higher Km (~175 nM) and but slightly higher Kcat (~8.2 sec-1). Now I have difficulty in understanding why is this happening. One more thing I observed is that S1 gets inhibited after 150 nM substrate concentration. While S2 continue to maintain it's saturation plateau even at 8000 nM. I am little perplexed how to rationalize this result. Any help will be appreciated much in this regard. Need an explanation about preferential degradation of the substrate by the enzyme.

Thank you in advance.

Prem

Hi, I'd like to try expression of reverse transcriptase with low temperature, 15 °C, due to solubility. Should I use some special plasmids for cold expression? I ordered cells for this purpose - E.coli Arctic express. Is it enough or is it better to combine these cells with plasmids for cold expression? Thank you all!

Hi guys,

Is there anybody who adds betaine (trimethylglycine) to media for protein expression? I read that betaine is able to increase solubility of proteins. If you use it for this purpose which form is the best? Is there any proven concentration?

Thank you for all answers!

Bohus

Hi, I'm trying to produce reverse transcriptase. I need to increase its solubility. I would be grateful for some experiences and advices. Thanks so much.

Hello!

I am looking for examples of industrial processes which are catalyzed by enzymes (either natural or recombinant). Particularly, I am interested in processes that end up with enzyme removal or inactivation (thermal or chemical), and I am trying to find examples from different areas - the food industry, textiles, pharmaceutics, fuel, etc.

Could you help, please?

First of all, I apologize for my poor English.

I would like to measure the enzymatic activity of COMT, referring to the following reference, but is it theoretically possible to substitute norepinephrine bitartrate hydrate?

Thank you very much.

Anyone know how to measure catalase activity in brain homogenates in terms of μmol/min/g of brain. Reaction was stopped by addition of dichromate and absorbance was measures after 1 min at 570 nm. Controls are without H2O2 for each samples.

Can you plzz provide some references for effect of Mn fertilization on soil enzymatic activities

Hi!

A special primer has to be used with only 19 bp poly dT (Tm 44.9) instead of 30 bp in original study (Tm 55.2). Will this induce failure of mRNA capture? Will this cause RNA detachment in Reverse transcription using Superscript II (@42 ℃)? If this is a problem, how about start reverse transcription @ 37℃ or 40℃ for 20min, then rise it to 42℃？ Will superscript II be completely block at lower temperature?

I have Laccase from trametes versicolor with 0.5U/mg activity.

I need enzymatic activity of 50U/g in solution. What should be the amount of enzyme I add to obtain enzymatic activity of the above said?

I am working with erythroid progenitor cells in culture and want to assess the activity of an intracellular (cytoplasmic) enzyme. I have a protocol from the literature for the activity assay, but I can't seem to find any detailed info on the lysis buffer and methods used prior to the assay. For westerns I lyse with RIPA on ice and then sonicate, but I know I can't use RIPA for this, so my two questions are:

1. What lysis buffer recipe do you recommend to extract cytoplasmic proteins for an activity assay?

2. What lysis method do you recommend in conjunction with this buffer? Details would be appreciated!

I know there are many lysis buffer options and I'm just not sure how to figure out which one is both easy to make and will work for this purpose, and if one lysis method (freeze/thaw vs sonication, etc.) is more/less suitable for retaining enzyme function for downstream testing?