Calculating Kcat from Specific activity?

Erome Daniel Hankore @Erome_Daniel_Hankore

06 June 2017 0 2K Report

[E]: 10 ug/mL

Vmax: 0.098 U/mg

Protein mw: 128 kDa

Reaction volume: 150 uL

I'm having trouble getting to S-1 units for Kcat determination.

Kcat = Vmax/[E] so I should divide 0.098 U/mg by 10 ug/mL of enzyme concentration right?

Badges
Science topic

More Erome Daniel Hankore's questions See All

Why are lines forming in resolving gel of SDS-PAGE and preventing separation of proteins?

This has really been quite frustrating. Every time I load my samples (20 to 45 ug per well), I see lines form in the resolving gel and they interrupt the run. The proteins just accumulate on that...

11 December 2019 7,305 10 View

Why is m9 medium turning yellow?

I prepared M9 medium by autoclaving the M9 salts and glucose separately. Calcium chloride, magnesium sulfate, and trace minerals were dissolved in autoclaved water and sterilized by filtration....

05 June 2018 4,688 4 View

Why do we put enzymes in ice after heating them for thermostability assays?

I am noticing that in thermostability assays call for heating the enzyme at the desired temp for a set time and then immediately placing in ice before carrying out the assay (as reported in...

02 March 2018 8,339 0 View

What is the best solvent to dissolve p-coumaric acid?

Any suggestions? Perhaps in NaOH and the neutralize to pH 7. I need the pH to be around 7 in the final solution.

01 February 2018 5,332 8 View

Substrate concentration lower than Km?

If I am using substrate concentration that is much lower than the reported Km of an enzyme, say for example 30x lower, is there a way to compensate for that? Can I just add an excess of enzyme...

31 December 2017 4,579 3 View

Two compounds in solution absorbing at the same wavlength?

So, I'm doing an enzyme assay and there are two compounds in the assay, one a product and the other a cofactor, that absorb at the same wavelength. It makes it very difficult for me to detect...

31 December 2017 3,510 3 View

How do you quench NADPH in enzyme assays?

What kind of chemical agents can I use to oxidize NADPH in a reaction and stop an enzymatic reaction? Suggestions other than chemical agents are also welcome. Thanks!

31 December 2017 1,601 5 View

How do you regulate antibiotic resistance gene expression?

When you have an antibiotic resistance gene in a plasmid, how do ensure that the enzyme is constitutively expressed so that your selection always works?

10 November 2017 5,123 5 View

Enzyme assays can't reach Vmax?

I am doing some enzyme assays with a non-native substrate of an enzyme. At a certain concentration, it seems to plateau (as if reaching Vmax) and the graph resembles MM kinetics but when I try...

07 August 2017 2,109 15 View

Is the best way to report enzyme activity Km and Kcat or specific activity?

I've been thinking a lot about this question of late. I suppose most people determine kinetic parameters in terms of Km and Kcat to see how well their substrate is catalyzed by an enzyme. But if a...

05 June 2017 4,984 11 View

Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence?

I am on the lookout for the Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence. Does anyone know where I can find it? Thank you in advance

03 March 2021 3,568 1 View

Can polymer concentrations be calculated based on the number average molecular weight?

Hello, I spin-cast two different polymers onto wafers and measure their height as a function of initial concentration. For polymer A, I have the weight-average molecular weight (Mw) as well as...

28 February 2021 7,882 2 View

What's the concentration recommend for recombinant protein use in western blotting?

I'm currently working having problem with the concentration for recombinant protein use in western blot, the one been used is Recombinant Human CD19 Fc Chimera Protein-rndsystem which does not...

24 February 2021 8,514 3 View

How to resolve Gaussian error termination " Error termination via Lnk1e in F:\G16W\l103.exe" for TS(QST3)?

When I run the job for TS(QST3) in gaseous phase, it was completed without any error. But when I add water in CPCM model it terminated with an error: Inconsistency: ModMin= 2 Eigenvalue=...

21 February 2021 3,198 2 View

Using the Pfaffl method: Is it possible that the ratio of the calibrator does not equal 1?

Hi all, I have a question regarding calculations using the Pfaffl method. I have 3 groups per experiment: 1 control group (calibrator) and 2 treatment groups with 6 to 7 biological repeats per...

19 February 2021 8,351 2 View

Will SARS-CoV-2 Spike be broken into S1 and S2 by denaturing conditions?

Hello, I am performing a western blot of my SARS-CoV-2 Spike-pseudotyped lentiviral particles. I denature my virus by adding concentrated virus to 2X Laemmli buffer at 1:1, then boiling for...

17 February 2021 4,844 4 View

Kinetics of Enzyme production in Recombinant E Coli?

Hello, I am trying to find kinetic information (such as rate of enzyme production etc.) on the expression of taq polymerase in E-Coli after inserting a recombinant plasmid. Any help would be...

12 February 2021 1,386 3 View

How to determinate of molecular structure of salt into the liquid?

I need to determine the open formula of our complex, presumably consisting of sodium tungstate Na2WO4 and aluminum sulfate Al2 (SO4) 3. Our problem is that with an increase in the concentration...

11 February 2021 5,821 1 View

During an anti-His capture ELISA has anyone had issues with histidine residues from secondary antibodies non-specifically binding?

Hi, Hope you can help. Currently been running some sandwich ELISA assays where I coat with anti-His, block and then capture His-tag protein and then sera and then anti-bovine monoclonal...

07 February 2021 7,101 1 View

Is it possible to quantify recombinant protein in the supernatant using SDS PAGE ?

I would like to quantify a recombinant protein (with native signal sequence) secreted by Pichia pastoris using the SDS PAGE. Since my standard protein (plant origin) is not pure, I would like to...

07 February 2021 3,880 9 View