Name: sigma's non-specific protease activity assay - casein as a substrate
Uploaded: 2008-09-17T09:14:00
Duration: 11 min 37 s
Description: Watch this Scientific Journal Video about Sigma's Non-specific Protease Activity Assay - Casein as a Substrate at JoVE.com

Before beginning the assay, we need to make sure that the following reagents are correctly prepared:
<ol>
<li>A 50 mM Potassium Phosphate Buffer, pH 7.5. Prepare using 11.4 mg/ml of potassium phospate dibasic, trihydrate in purified water and adjusting pH with 1M HCl. This solution is placed at 37&deg;C prior to use.</li>
<li>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 &deg;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. </li>
<li>A 110 mM Trichloroacetic acid solution, prepared by diluting a 6.1N stock 1:55 with purified water. Trichloroacetic acid is a strong acid and should be handled with care.</li>
<li>0.5 mM Folin &amp; Ciolcaltea's, or Folin's Phenol Reagent, which is the solution that will react with tyrosine to generate a measurable color change that will be directly related to the activity of proteases. Folin's Phenol Reagent is an acid and should be handled with care. </li>
<li>A 500 mM Sodium Carbonate solution, prepared using 53 mg/ml of anyhydrous sodium carbonate in purified water. An enzyme diluent solution, which consists of 10 mM Sodium Acetate Buffer with 5mM Calcium Acetate, pH 7.5, at 37&deg;C. This solution is what we use to dissolve solid protease samples or dilute enzyme solutions.</li>
<li>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.</li>
<li>Protease solution. Immediately before use, dissolve protease in enzyme diluent solution prepared in step 6.</li>
</ol>
If necessary, use a solid protease sample of predetermined activity, which is dissolved using enzyme diluent to 0.1-0.2 units/ml. This solution serves as a positive control for the quality control assay and as validation for the calculations we will perform to determine enzyme activity.
Setting up the Protease Assay and Standard Curves
<ol>
<li>To begin this assay, find suitable vials that will hold about 15 mls. For each enzyme that will be tested, 4 vials are needed. One vial will be used as a blank, and three others will be used to assay activity of three dilutions of the protease. Three dilutions are useful when checking final calculations against each other. <ol>
<li>To each set of four vials, add 5mls of our 0.65% casein solution. Let them equilibrate in a water bath at 37&deg;C for about 5 minutes. </li>
<li>Add varying volumes of enzyme solution that will be tested to three of the test sample vials, but not the blank. Mix by swirling and incubate for 37&deg;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.</li>
</ol></li>
<li>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. Incubate the solutions at 37&deg;C for 30 minutes. During this 30 minute incubation, you may want to set up your tyrosine standard dilutions. 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.</li>
<li>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. <ol>
<li>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. </li>
<li>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. </li>
<li>Add sodium carbonate to regulate any pH drop created by the addition of the Folin's reagent.</li>
<li>Add sodium carbonate to the test samples and test blank. These solutions become cloudy after the addition of sodium carbonate. Add the Folin's reagent, which will react primarily with free tyrosine. </li>
<li>Mix the dram vials by swirling and incubate at 37&deg;C for 30 minutes.</li>
</ol>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 in our test samples. 2mls of these solutions are filtered using a 0.45 um polyethersulfone syringe filter into suitable cuvettes. Now that the assay is performed,&nbsp;you can proceed to the spectrophotometer to record our absorbance values. </li>
</ol>
Measuring Absorbance and Calculating Enzyme Activity
<ol>
<li>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. After this simple calculation, create the standard curve using a graphing program to plot the change in absorbance of our standards on the Y axis, versus the amount in micromoles for each of our 5 standards on the X axis. 
<table style="width: 416px; height: 118px;" border="0">
<tbody>
<tr>
<td style="text-align:center">Volume of Tyrosine Standard </td><td style="text-align:center">uMoles Tyrosine </td>
</tr>
<tr>
<td style="text-align:center">0.05</td>
<td style="text-align:center">0.055</td>
</tr>
<tr>
<td style="text-align:center">0.10</td>
<td style="text-align:center">0.111</td>
</tr>
<tr>
<td style="text-align:center">0.20</td>
<td style="text-align:center">0.221</td>
</tr>
<tr>
<td style="text-align:center">0.40</td>
<td style="text-align:center">0.442</td>
</tr>
<tr>
<td style="text-align:center">0.50</td>
<td style="text-align:center">0.553</td>
</tr>
</tbody>
</table>
</li>
<li>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 assay 10= Time of assay (in minutes) as per the Unit definition 1= Volume of Enzyme (in milliliters) of enzyme used 2= Volume (in milliliters) used in Colorimetric Determination Take 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 our case is 11mls. Divide this value by three other quantities: the time of the assay, which we 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. We used 2 mls.</li>
<li>Micromoles of tyrosine divided by time in minutes yields measurement of protease activity called "units". We can cancel out the units for volume measurement in the numerator and denominator, leaving a measurment of enzyme activity in terms of units/ml. In order to determine the activity in a solid protease sample diluted in enzyme diluent, we divide our activity in units/ml by the concentration of solid used in this assay originally in mg/ml., leaving us with activity in terms of units/mg. Units/ml enzyme Units/mg solid = _____________________ mg solid/ ml enzyme </li>
</ol>

<ol>
	<li>Anson, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J. Gen. Physiol. 22, 79-89 (1938).</li><li>Folin, O., Ciocalteau, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J. Biol. Chem. 73, 627 (1929).</li></ol>

We've just shown you how to analyze protease activity using Sigma's universal protease activity assay. In addition, this assay is useful to ensure that our proteases have precisely determined activity before you receive them for your experiments. As you have seen, when doing this procedure, it's of paramount importance to remember to heat both the casein and tyrosine solutions slowly and not to boil them. Also, it's critical to prepare different blanks for both your standards and for each test sample that you have.

<table>
	<tbody>
		<tr>
			<td>Protease</td>
			<td>Sigma-Aldrich</td>
			<td>P4630</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate, Dibasic, Trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>P5504</td>
			<td></td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma-Aldrich</td>
			<td>C7078</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>T0699</td>
			<td></td>
		</tr>
		<tr>
			<td>Folin &#38; Ciocalteu&#39;s Phenol Reagent</td>
			<td>Sigma-Aldrich</td>
			<td>F9252</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Carbonate, Anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>S2127</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Acetate, Trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S8625</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>C1000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tyrosine, Free Base</td>
			<td>Sigma-Aldrich</td>
			<td>T3754</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Profiler Kit</td>
			<td>Sigma-Aldrich</td>
			<td>PP0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Fluorescent Detection Kit</td>
			<td>Sigma-Aldrich</td>
			<td>PF0100</td>
			<td></td>
		</tr>
	</tbody>
</table>

sigma's non-specific protease activity assay - casein as a substrate

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases. Sigma's non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases, which is what we do during our quality control procedures. In this assay, casein acts as a substrate. When the protease we are testing digests casein, the amino acid tyrosine is liberated along with other amino acids and peptide fragments. Folin and Ciocalteus Phenol, or Folin's reagent primarily reacts with free tyrosine to produce a blue colored chromophore, which is quantifiable and measured as an absorbance value on the spectrophotometer. The more tyrosine that is released from casein, the more the chromophores are generated and the stronger the activity of the protease. Absorbance values generated by the activity of the protease are compared to a standard curve, which is generated by reacting known quantities of tyrosine with the F-C reagent to correlate changes in absorbance with the amount of tyrosine in micromoles. From the standard curve the activity of protease samples can be determined in terms of Units, which is the amount in micromoles of tyrosine equivalents released from casein per minute.
To view this article in Chinese, <a href="http://jove.com/index/details.stp?ID=910">click here</a>

Watch this Scientific Journal Video about Sigma's Non-specific Protease Activity Assay - Casein as a Substrate at JoVE.com

Sigma's Non-specific Protease Activity Assay - Casein as a Substrate

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases. Sigma's non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases.

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases.  Sigma's non-specific protease ...

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Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

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Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
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An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

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A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

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Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes1-3. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of 18O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao4, Miyagi and Rao5 and Ye et al.6). 18O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques7. Depending on the protease utilized, 18O-labeling can result in the incorporation of up to two 18O-atoms in the C-terminal carboxyl group of the cleavage product3. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction8. In our PALeO (protease-assisted labeling employing 18O-enriched water) adaptation of enzymatic 18O-labeling, we utilized 50% 18O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases9 and monitor proteolytic reactions10-11. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second 18O-atom. Such "double-labeling" enzymes can be used for postdigestion 18O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing 18O-enriched water beyond enzymes and uses acidic pH conditions to introduce 18O-stable isotope signatures into peptides.

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Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...