At the last formula which calculates Solid protease sample Activity, using the activity(units/ml) from the previous formula and divide it by the concentration of solid. My question is in the calculation result shows in the same activity(units/ml), the higher solid concentration i use, the lower activity I get? eg: in the video use 0.275(units/ml) divided by 0.015(mg/ml) equals to 18.3 units/mg, but id use higher solid concentration like 0.030, you get 0.275(units/ml) divided by 0.030(mg/ml) equals to 9.16 units/mg, which shows higher solid concentration gets lower solid activity. I'm really confuse about it and need a explanation to it.
I don’t have protease enzyme solution to make blank for test. Then what should I do for it? Can i take distilled water instead of enzyme solution and proceed the protocol?
The absorbance of the samples is measured by a spectrophotometer using a wavelength of 660nm. The light path is set to 1cm. Record the absorbance values for the standards, standard blank, the different test samples, and test blank. Once all of the data has been collected, the standard curve can be created. In order to generate the curve, difference in absorbance between the standard and standard blank must be calculated. This is the absorbance value attributable to the amount of tyrosine in the standard solutions.
Thanks for your video. I would like to use azocasein instead of casein, can I get more information about it? I heard that azocasein is more accurate than casein.
Both are good except that in azocasein, it functions by releasing the azo-dye for absorbance detection. Casein operates by releasing aromatic amino acid residues which react with Folin's reagent to form chromophoric products
Question on the F-C Reagent. It comes as a 2N solution but this protocol calls for a 0.5 mM solution. What is used as the diluent and how does 2N F-C reagent correlate to 0.5 mM F-C reagent?
Sorry, it is 0.5 M Folin & Ciolcaltea’s not mM, then You use this formula C1V1=C2V2. Molarity is equal to Normality and you just need to take 1 ml of the commercial reagent and fill it up with deionized water to a total volume of 4 ml
Here is it: After data points have been entered, generate a line of best fit and corresponding slope equation.Find the change in absorbance in the test samples by calculating the difference between the test sample absorbance and the absorbance of the test blank. Inserting the absorbance value for one of the test samples into the slope equation and solving will result in the micromoles of tyrosine liberated during this particular proteolytic reaction. To get the activity of enzyme in units per/ml, perform the following calculation:(umole tyrosine equivalents released) x (11) Units/ml Enzyme = __________________________________________(1) x (10) x (2)11= Total volume (in milliliters) of assay10= Time of assay (in minutes) as per the Unit definition1= Volume of Enzyme (in milliliters) of enzyme used2= Volume (in milliliters) used in Colorimetric DeterminationTake the number of micromoles tyrosine equivalents released obtained from the slope equation and multiply it by the total volume of the assay in mls, which in this case is 11mls. Divide this value by three other quantities: the time of the assay, which here ran for 10 minutes, the volume of enzyme used in the assay, which was varied (let's use 1ml), the volume of milliliters used in colorimetric detection, which may differ based on your cuvette. Here it is 2 mls.
@@Educationalcourses I am asking your slope of standard curve? I get 0.008 slope of standard curve and once get 0.002 so which one is correct so I want to know your slope.
@@yatrimehta381 As I remember it could vary depending on the type of the sample. So, all you should concentrate on is that your sample readings aren't outside the linear range.
No, you have to incubate the samples for exactly 10 min, because you will measure protease activity and consequential liberation of tyrosine in this incubation time so it will affect the calculation.
you need a suitable concentration of the enzyme not too low and be careful that temperature and PH affect protease concentration so you may experience very low concentration because of the temperature or the PH.
Thank you for the response. I determined my crude protease is the highest activity at 60 C and pH 9. So can I incubate the enzyme-casein mixture on these conditions?
If you examined at various temperatures by incubating enzyme reaction mixture at different temperatures and this what you got so, you can incubate the mixture in these condition.
I incubated at those conditions (60C and pH 9). But the absorbance values of the blank sample are too high between 1.7-1.8 abs. I couldn’t figure out the reason. I did prepared the blank as in the video (5 mL casein solution was incubated at 37C, 5 min. I didn’t add any enzyme solution into the casein. I incubated all the samples at the above conditions. 5 mL TCA was added, and then I added 1 mL of enzyme for the blank. Following steps are incubation 37C, 30 min.) and filtration. 2 mL filtrate was mixed with 5 mL sodium carbonate and 1 mL FCR. After incubating at 37C for 30 min absorbance values were measured at 660 nm. Which point I do wrongly?
@@Educationalcoursesbut we add tca in the standard curve which inhibits the formation of color so we not understand it.( if add TCA in Standard it's give yellow color and after 24hr show blue color so I think TCA effect on the FCR- tyrosine reaction)
@@yatrimehta381 I am really uncertain about the exact reason behind the outcome you are experiencing, but I can provide you with the protocol for setting up standard curves. 1)To each set of four vials, add 5mls of our 0.65% casein solution. Let them equilibrate in a water bath at 37°C for about 5 minutes. 2)Add varying volumes of enzyme solution that will be tested to three of the test sample vials, but not the blank. 3)Mix by swirling and incubate for 37°C for exactly ten minutes. The protease activity and consequential liberation of tyrosine during this incubation time is what will be measured and compared between test samples. 4)After this 10 minute incubation, add the 5 mls of the TCA reagent to each tube to stop the reaction. Then, add an appropriate volume of enzyme solution to each tube, even the blank, so that the final volume of enzyme solution in each tube is 1 ml. This is done to account for the absorbance value of the enzyme itself and to ensure that the final volume in each tube is equal. 5)Incubate the solutions at 37°C for 30 minutes.During this 30 minute incubation, you may want to set up your tyrosine standard dilutions. 6)Use 6 dram vials (dram vials can be substituted with polypropylene tubes) that can easily hold 8 mls. To the six vials, add the 1.1 mM tyrosine standard stock solutions with the following volumes in mls: 0.05, 0.10, 0.20, 0.40, 0.50. Don't add any tyrosine standard to the blank. Lower standards may be needed for impure test samples that will yield little color change. Once the tyrosine standard solution has been added, add an appropriate volume of purified water to each of the standards to bring the volume to 2 mls. 7)After the 30 minute incubation, filter each of the test solutions and the blank using a 0.45 um polyethersulfone syringe filter. Filtration is required to remove any insolubles from the samples. 8)Add the filtration 2 mls of the test samples and blank filtrate to 4 dram vials that can hold at least 8 mls. The same type of vial in which the standards were prepared can be used. 9)To all of the vials containing the standards and standard blank, add 5mls of sodium carbonate. For best results, add 1 ml of Folin's reagent immediately afterwards. Add sodium carbonate to regulate any pH drop created by the addition of the Folin's reagent. Add sodium carbonate to the test samples and test blank. These solutions become cloudy after the addition of sodium carbonate. 10)Add the Folin's reagent, which will react primarily with free tyrosine. Mix the dram vials by swirling and incubate at 37°C for 30 minutes. 11)After this incubation, you should notice that the standards have a gradation of color correlating with the amount of tyrosine added; the highest concentrations of tyrosine appearing darkest. You can also notice appreciable color change. 2mls of these solutions are filtered using a 0.45 um polyethersulfone syringe filter into suitable cuvettes. 12)Now that the assay is performed, you can proceed to the spectrophotometer to record our absorbance values.
For casein: A 0.65% weight/volume casein solution, prepared by mixing 6.5 mg/ml of casein in the 50 mM potassium phosphate buffer. Gradually increased the solution temperature with gentle stirring to 80-85 °C for about 10 minutes until a homogenous dispersion is achieved. It is very important not to boil the solution. The pH is then adjusted if necessary with NaOH and HCl. For tyrosine: 1.1 mM L-tyrosine Standard stock solution. Prepared using 0.2 mg/ml L-tyrosine in purified water and heated gently until the tyrosine dissolves. As with the casein, do not boil this solution. Allow the L-tyrosine standard to cool to room temperature. This solution will be diluted further to make our standard curve.
well, proteolytic activity can be measured by different methods using several substrates. For using casein to quantify the enzyme activity, I recommend this study link.springer.com/article/10.1007/s11274-014-1598-z and this www.ncbi.nlm.nih.gov/pmc/articles/PMC2929773/
Use 6 dram vials (dram vials can be substituted with polypropylene tubes) that can easily hold 8 mls. To the six vials, add the 1.1 mM tyrosine standard stock solutions with the following volumes in mls: 0.05, 0.10, 0.20, 0.40, 0.50. Don't add any tyrosine standard to the blank. Lower standards may be needed for impure test samples that will yield little color change. Once the tyrosine standard solution has been added, add an appropriate volume of purified water to each of the standards to bring the volume to 2 mls.
At the last formula which calculates Solid protease sample Activity, using the activity(units/ml) from the previous formula and divide it by the concentration of solid. My question is in the calculation result shows in the same activity(units/ml), the higher solid concentration i use, the lower activity I get? eg: in the video use 0.275(units/ml) divided by 0.015(mg/ml) equals to 18.3 units/mg, but id use higher solid concentration like 0.030, you get 0.275(units/ml) divided by 0.030(mg/ml) equals to 9.16 units/mg, which shows higher solid concentration gets lower solid activity. I'm really confuse about it and need a explanation to it.
I don’t have protease enzyme solution to make blank for test. Then what should I do for it?
Can i take distilled water instead of enzyme solution and proceed the protocol?
Question on the mesaturing.how how exactly to measure absorbance
The absorbance of the samples is measured by a spectrophotometer using a wavelength of 660nm. The light path is set to 1cm. Record the absorbance values for the standards, standard blank, the different test samples, and test blank. Once all of the data has been collected, the standard curve can be created. In order to generate the curve, difference in absorbance between the standard and standard blank must be calculated. This is the absorbance value attributable to the amount of tyrosine in the standard solutions.
Thanks for your video.
I would like to use azocasein instead of casein, can I get more information about it?
I heard that azocasein is more accurate than casein.
Both are good except that in azocasein, it functions by releasing the azo-dye for absorbance detection. Casein operates by releasing aromatic amino acid residues which react with Folin's reagent to form chromophoric products
Question on the F-C Reagent. It comes as a 2N solution but this protocol calls for a 0.5 mM solution. What is used as the diluent and how does 2N F-C reagent correlate to 0.5 mM F-C reagent?
Sorry, it is 0.5 M Folin & Ciolcaltea’s not mM, then You use this formula C1V1=C2V2. Molarity is equal to Normality and you just need to take 1 ml of the commercial reagent and fill it up with deionized water to a total volume of 4 ml
tyrosine is aromatic amino acid so we should take OD at 280 nm..?
Please reply..
At the formula for activity calculation , why we should divide with the volume of sample put in cuvet ( eg. 2 mL like in the video)?
This may differ based on your cuvette
And if possible please tell tyrosine standard curve slope..? Because I try many times and got different results.
Here is it: After data points have been entered, generate a line of best fit and corresponding slope equation.Find the change in absorbance in the test samples by calculating the difference between the test sample absorbance and the absorbance of the test blank. Inserting the absorbance value for one of the test samples into the slope equation and solving will result in the micromoles of tyrosine liberated during this particular proteolytic reaction. To get the activity of enzyme in units per/ml, perform the following calculation:(umole tyrosine equivalents released) x (11) Units/ml Enzyme = __________________________________________(1) x (10) x (2)11= Total volume (in milliliters) of assay10= Time of assay (in minutes) as per the Unit definition1= Volume of Enzyme (in milliliters) of enzyme used2= Volume (in milliliters) used in Colorimetric DeterminationTake the number of micromoles tyrosine equivalents released obtained from the slope equation and multiply it by the total volume of the assay in mls, which in this case is 11mls. Divide this value by three other quantities: the time of the assay, which here ran for 10 minutes, the volume of enzyme used in the assay, which was varied (let's use 1ml), the volume of milliliters used in colorimetric detection, which may differ based on your cuvette. Here it is 2 mls.
@@Educationalcourses I am asking your slope of standard curve? I get 0.008 slope of standard curve and once get 0.002 so which one is correct so I want to know your slope.
@@yatrimehta381 I think it is .8 but this is at 660nm
@@Educationalcourses means I get 0.008 standard slope which is not correct(I also take it at 660nm) ? I should get 0.8 right?
@@yatrimehta381 As I remember it could vary depending on the type of the sample. So, all you should concentrate on is that your sample readings aren't outside the linear range.
Can the sample be incubated for more than 10min or will that effect the calculation? What If the sample has very low concentration of enzyme?
No, you have to incubate the samples for exactly 10 min, because you will measure protease activity and consequential liberation of tyrosine in this incubation time so it will affect the calculation.
you need a suitable concentration of the enzyme not too low and be careful that temperature and PH affect protease concentration so you may experience very low concentration because of the temperature or the PH.
How can we estimate the protease activity of bacteria
I think this would help you: www.ncbi.nlm.nih.gov/pmc/articles/PMC10062638/
How long should we keep the standard samples at 37 C after dilute the Tyrosine to a total volume of 2 mL?
Incubate the samples for 30 minute
Thank you for the response. I determined my crude protease is the highest activity at 60 C and pH 9. So can I incubate the enzyme-casein mixture on these conditions?
If you examined at various temperatures by incubating enzyme reaction mixture at different temperatures and this what you got so, you can incubate the mixture in these condition.
And the same thing for pH
I incubated at those conditions (60C and pH 9). But the absorbance values of the blank sample are too high between 1.7-1.8 abs. I couldn’t figure out the reason. I did prepared the blank as in the video (5 mL casein solution was incubated at 37C, 5 min. I didn’t add any enzyme solution into the casein. I incubated all the samples at the above conditions. 5 mL TCA was added, and then I added 1 mL of enzyme for the blank. Following steps are incubation 37C, 30 min.) and filtration. 2 mL filtrate was mixed with 5 mL sodium carbonate and 1 mL FCR. After incubating at 37C for 30 min absorbance values were measured at 660 nm. Which point I do wrongly?
Should add TCA in standard reading?
We add TCA to stop the reaction after that we filter each of the test solutions and the blank using a 0.45 um polyethersulfone syringe filter.
@@Educationalcoursesbut we add tca in the standard curve which inhibits the formation of color so we not understand it.( if add TCA in Standard it's give yellow color and after 24hr show blue color so I think TCA effect on the FCR- tyrosine reaction)
@@yatrimehta381 I am really uncertain about the exact reason behind the outcome you are experiencing, but I can provide you with the protocol for setting up standard curves.
1)To each set of four vials, add 5mls of our 0.65% casein solution. Let them equilibrate in a water bath at 37°C for about 5 minutes.
2)Add varying volumes of enzyme solution that will be tested to three of the test sample vials, but not the blank. 3)Mix by swirling and incubate for 37°C for exactly ten minutes. The protease activity and consequential liberation of tyrosine during this incubation time is what will be measured and compared between test samples.
4)After this 10 minute incubation, add the 5 mls of the TCA reagent to each tube to stop the reaction. Then, add an appropriate volume of enzyme solution to each tube, even the blank, so that the final volume of enzyme solution in each tube is 1 ml. This is done to account for the absorbance value of the enzyme itself and to ensure that the final volume in each tube is equal.
5)Incubate the solutions at 37°C for 30 minutes.During this 30 minute incubation, you may want to set up your tyrosine standard dilutions.
6)Use 6 dram vials (dram vials can be substituted with polypropylene tubes) that can easily hold 8 mls. To the six vials, add the 1.1 mM tyrosine standard stock solutions with the following volumes in mls: 0.05, 0.10, 0.20, 0.40, 0.50. Don't add any tyrosine standard to the blank. Lower standards may be needed for impure test samples that will yield little color change. Once the tyrosine standard solution has been added, add an appropriate volume of purified water to each of the standards to bring the volume to 2 mls.
7)After the 30 minute incubation, filter each of the test solutions and the blank using a 0.45 um polyethersulfone syringe filter. Filtration is required to remove any insolubles from the samples.
8)Add the filtration 2 mls of the test samples and blank filtrate to 4 dram vials that can hold at least 8 mls. The same type of vial in which the standards were prepared can be used.
9)To all of the vials containing the standards and standard blank, add 5mls of sodium carbonate. For best results, add 1 ml of Folin's reagent immediately afterwards.
Add sodium carbonate to regulate any pH drop created by the addition of the Folin's reagent.
Add sodium carbonate to the test samples and test blank. These solutions become cloudy after the addition of sodium carbonate.
10)Add the Folin's reagent, which will react primarily with free tyrosine.
Mix the dram vials by swirling and incubate at 37°C for 30 minutes.
11)After this incubation, you should notice that the standards have a gradation of color correlating with the amount of tyrosine added; the highest concentrations of tyrosine appearing darkest. You can also notice appreciable color change. 2mls of these solutions are filtered using a 0.45 um polyethersulfone syringe filter into suitable cuvettes.
12)Now that the assay is performed, you can proceed to the spectrophotometer to record our absorbance values.
For the enzyme diluent what is used for a calcium source?
The enzyme diluent solution consists of 10 mM Sodium Acetate Buffer with 5mM Calcium Acetate, pH 7.5, at 37°C
How enzyme solution was prepared
For casein: A 0.65% weight/volume casein solution, prepared by mixing 6.5 mg/ml of casein in the 50 mM potassium phosphate buffer. Gradually increased the solution temperature with gentle stirring to 80-85 °C for about 10 minutes until a homogenous dispersion is achieved. It is very important not to boil the solution. The pH is then adjusted if necessary with NaOH and HCl.
For tyrosine: 1.1 mM L-tyrosine Standard stock solution. Prepared using 0.2 mg/ml L-tyrosine in purified water and heated gently until the tyrosine dissolves. As with the casein, do not boil this solution. Allow the L-tyrosine standard to cool to room temperature. This solution will be diluted further to make our standard curve.
nice video..
how can i estimate protease of fungus ?
well, proteolytic activity can be measured by different methods using several substrates. For using casein to quantify the enzyme activity, I recommend this study
link.springer.com/article/10.1007/s11274-014-1598-z and this
www.ncbi.nlm.nih.gov/pmc/articles/PMC2929773/
The protocol in this video is universal, I think you can use the same steps to estimate protease activity
For the enzyme diluent should the buffers be mixed at 1:1 ratio?
Use 6 dram vials (dram vials can be substituted with polypropylene tubes) that can easily hold 8 mls. To the six vials, add the 1.1 mM tyrosine standard stock solutions with the following volumes in mls: 0.05, 0.10, 0.20, 0.40, 0.50. Don't add any tyrosine standard to the blank. Lower standards may be needed for impure test samples that will yield little color change. Once the tyrosine standard solution has been added, add an appropriate volume of purified water to each of the standards to bring the volume to 2 mls.