Beacon Protease Activity Detection Kit Protocol

Transcription

1 Part # P2010 Lit. # L0054 Rev. 03/01 Page 1 of INTENDED USE The Beacon 2000 Protease Activity Detection Kit is designed to determine protease activity in biological samples using fluorescence polarization (FP). The Fluorescence Polarization (FP)-based protease assay is extremely sensitive, does not rely on radioactivity, and requires no separation to acquire quantitative results. The sample is simply incubated with the Beacon Fluorescein-succinimidyl estercasein (FSE casein) substrate in the supplied incubation buffer and the extent of substrate degradation is then measured using the Beacon 2000 Analyzer. The general reaction scheme is as follows: Protease FSE-casein FSE-peptides (high polarization) (low polarization) Protease activity in the sample cleaves FSE-casein to smaller FSE-peptides. The protease activity is then measured by a proportional decrease in fluorescent polarization. Polarization (P) is a ratio of intensities and is therefore a dimensionless number. The Beacon 2000 System reports polarization values in millipolarization (mp). 2.0 SAFETY PRECAUTIONS Normal precautions exercised in handling laboratory reagents should be followed. The reagents supplied are not considered hazardous according to 29 CFR However, the Beacon Incubation Buffer and Beacon Assay Buffer are considered irritants. The chemical, physical, and toxicological properties of these products may not, as yet, have been thoroughly investigated. We recommend the use of gloves, lab coats, and eye protection when working with any chemical reagents. 3.0 DESCRIPTION 3.1 Materials Supplied The Beacon 2000 Protease Activity Detection Kit contains reagents sufficient for at least 200 assays. Description Composition Amount Part No. Beacon Fluoroscein-succinimidyl ester-casein (FSE-casein) 2 µg/ml in Beacon Incubation Buffer and 0.1% Triton 5 1 ml P2820 Beacon Incubation Buffer 50 mm Tris-HCl (ph 7.8), 150 mm NaCl, 0.02% NaN ml P2089 Beacon Assay Buffer 0.2 M Tris (ph 9.0), 0.5% SDS, 0.02% NaN ml P2064 Beacon Protease Positive Control 1 mg/ml Pronase E in PBS, 10 mg/ml BSA 1 ml P Materials Required but Not Supplied Trichloroacetic Acid (TCA), 60% Beacon 2000 Analyzer (equipped with a 488 nm excitation filter and a 535 nm emission filter) Pipetting devices for accurate delivery of volumes required for the assay Disposable 10 x 75 mm borosilicate test tubes, certified for use with the Beacon 2000 Analyzer (Part No. P2245) Autoclaved 0.5 ml or 1.5 ml microfuge tubes Laboratory timer 3.3 Storage and Stability The FSE-casein should be stored at 20 C and thawed on ice. Multiple freeze-thaw cycles may lead to a slight decrease in the starting polarization value of the substrate. FSE-casein is light-sensitive and should be protected from light during storage. The Protease Positive Control should be stored at 20 C. As with many proteases, the positive control is susceptible to autocleavage and there may be a small reduction in activity with time. The Incubation and Assay Buffers may be stored at 4 C or room temperature. All components are stable for 6 months from the date of purchase.

2 Part # P2010 Lit. # L0054 Rev. 03/01 Page 2 of Kit Performance This kit can be used to identify protease contamination, quantitate protease activity, or measure enzymatic kinetics of a protease sample. The End-Point assay will work in identifying protease contamination and provide a relative quantitation of activity. Precise quantitation of specific protease activity requires the construction of a standard curve using the protease of interest. In a 1 hour reaction at 37 C, typical limits of detection of this kit are: 0.18 U Trypsin, Bovine Pancreas U Chymotrypsin, Bovine Pancreas 0.09 U Thermolysin, Bacillus thermoproteolyticus 0.08 U Cathepsin B, Bovine Spleen The Trypsin, Chymotrypsin, and Thermolysin were diluted in 10 mm Tris-HCl, ph 7.8 prior to assay. The Cathepsin B was diluted in 10 mm Sodium Acetate, ph 5.2, 4 mm EDTA, and 5 mm Cysteine prior to assay. The detection limit was defined as the amount of enzyme required to cause a 20% change in fluorescence polarization compared to the control value. The kit will have different sensitivities to different protease species. The assay sensitivity may be increased by lengthening the reaction time to 18 hours (overnight) with minimal autodegradation of the substrate. It may be possible to substitute your own specific reaction buffer for the assay, but you will need to verify the background fluorescence prior to executing the reaction. Instructions for verifying your buffer are included in Section 4.1. Refer to the Beacon 2000 Instruction manual for detailed procedures for creating and editing Beacon protocols. 4.0 PROCEDURE 4.1 Experimental Design To identify protease activity in your sample, perform an Endpoint Assay (see Section 4.2). Each set of assays should include a buffer blank tube (incubation buffer/water only), a negative control tube (substrate + incubation buffer/water), a positive control (substrate, incubation buffer/water, protease positive control) and sample tubes (substrate, incubation buffer/water, sample). The buffer blank provides a sample for blanking the Beacon 2000 as well as to provide a zero to compare background fluorescence of the experimental sample. The negative control provides the starting (undigested) polarization data point. A second negative control may be constructed (immediately prior to measuring fluorescent polarization) whenever prolonged incubations (>4 hours) are performed. This second negative control, when compared to the incubated negative control, will indicate the degree, if any, of autodegradation not associated with the unknown sample. The positive control will indicate the degree of total degradation of the FSE-casein. The sample tubes will contain substrate and the sample. Standard reaction conditions are 1 hour at 37 C. Changing the reaction temperature to the optimal temperature for the target protease (if known) may increase the sensitivity of the assay. Increasing the reaction time up to 18 hours (overnight) may also increase the sensitivity, but may not be proportional due to inherent instability of some proteases. The Beacon 2000 Protease Activity Detection Kit contains an Incubation Buffer with a ph of 7.8. This ph is recommended for detecting the broadest range of physiological protease activities (e.g., ph ). For most physiological applications, the supplied Incubation Buffer will yield useful protease activity information. However, if a specific enzyme with a unique optimal ph is a suspected contaminant, or if ph activity curves will be generated, we suggest preparing ph-specific buffers. Detecting protease activity using this kit does not require generating a standard curve. When testing crude samples containing one or more unknown proteases, a standard curve would have little meaning. We suggest relating protease activity to changes in polarization per unit volume or mass of the sample (e.g., mp/total protein concentration). If necessary, compare purified protease preparations to an appropriate standard of known specific activity. Care must be taken when interpreting standard enzyme activity curves because enzyme activity can vary greatly depending on storage conditions, freeze-thaw cycles, and incubation buffers. The Protease Positive Control included with this kit is not appropriate for use as a standard, but may be used as a reference point. Running a Static Mode protocol on the Beacon Analyzer, use the buffer blank to account for fluorescence background in the buffer. Continue to measure the polarization of the control and sample tubes. Any decrease in polarization in the sample tubes compared to the negative control tube denotes the presence of protease. A useful way to report these data is mp/unit of sample. Accounting for fluorescence background in your protease sample may be necessary if the fluorescence background of the sample is greater than 10% to 20% of the FSE-casein intensity. If you are uncertain about the inherent fluorescence of your sample include a separate blank tube that contains sample and no FSE-casein as well as a buffer blank that has no FSE-casein or sample. This modified procedure is described in detail in Section 4.2.E.

3 Part # P2010 Lit. # L0054 Rev. 03/01 Page 3 of 7 The Beacon 2000 Protease Assay can be used to measure protease reaction kinetics. By using the temperature control feature of the Beacon 2000 and the supplied incubation buffer (or your specialized buffer), the kinetic activity of your protease sample can be measured. The controls and methods are described in Section Endpoint Assay Procedure A. Create a Beacon Protocol 1. Please refer to the Beacon 2000 Operator's Manual for detailed information regarding the operation of the Beacon 2000 Analyzer. Turn on the Beacon 2000 Analyzer. The instrument will perform a diagnostic procedure and produce a warning message if the RS- 232 port or the printer are not online. Press ENT to acknowledge any RS-232 or Printer not connected warnings, as these devices must be brought online before you can send data to them. The instrument will then display the first item of the Main Menu 1) Run Protocol. Allow the instrument to warm up for at least 20 minutes before further use. 2. If this protocol has been programmed into the Beacon 2000 previously, proceed to step B. Always confirm that the protocol printed on the thermal paper printout is the correct protocol selected. The general procedure for creating a Beacon protocol is as follows: 3. Using the UP and DWN keys, toggle to the Create Protocol option and press the ENT key. The Beacon 2000 will report the number of open protocol numbers. Press ENT again. 4. For the Protocol ID #, type 0 and 3 (or any number between 03 and 99 that has not already been defined). Press ENT. 5. Enter a 4-digit password using the numerical part of the instrument keypad and press ENT. 6. From the Select Read Mode menu use the UP and DWN keys to select Static and then press ENT. From the Select Blank Type menu, select Single Blank, and then press ENT to input a default Blank Delay of 0 seconds. 7. Press ENT to input a default Sample Delay of 0 seconds. 8. At the Number of Average Read Cycles prompt, type 3, then ENT. 9. Press ENT to select the default Set Temperature setting and press 2 and 5 to set the chamber temperature to 25 C. 10. Press ENT two more times to select the default Auto Range and Printer options. You may review a hard copy of the protocol by pressing YES to the Print Protocol prompt. 11. If you have created the protocol correctly, press YES, at the Store New Protocol? prompt and the protocol will be saved under the designated Protocol ID number. B. Prepare Reagents 1. Determine how many reactions are needed. Each set of assays should include one positive, one negative, and one Buffer Blank control tubes. The sample tubes will contain up to 100 µl of your sample. Single tube reaction recipes are described in following Table: Reagent Buffer Blank Sample Blank (Optional) Positive Control Negative Control Samples Sample µl µl Incubation Buffer 150 µl 150 µl 150 µl 150 µl 150 µl Protease Free Water 125 µl 0-99 µl 90 µl 100 µl 0 99 µl Protease Positive Control µl FSE-Casein µl 25 µl 25 µl 2. Combine the reagents in 0.5 ml or 1.5 ml microfuge tubes. Whenever possible it is recommended that you set up your assay from a Master Mix (except for the Buffer Blank ). A recipe for a Master Mix can be calculated from the volumes per reaction used in the negative control. When making this Master Mix, you will need to account for the sample volumes to be tested and a little extra to allow for accurate pipeting. For example, if you are testing 10, 20 and 30 µl of a single reagent set up the reaction using the following Master Mix for 5.5 Reactions (1 Positive Control, 1 Negative Control, 3 Samples, plus 0.5 reaction to allow for accurate pipeting): 825 µl Incubation Buffer 385 µl Protease-Free Water µl FSE-casein Aliquot 245 µl of the Master Mix into each of 5 nuclease-free microfuge tubes. Bring up the volume to 275 µl per tube with water (negative control), 10 µl protease positive control + 20 µl water (positive control), or test sample/water.

4 Part # P2010 Lit. # L0054 Rev. 03/01 Page 4 of 7 C. Perform the Assay 1. Standard reaction conditions are 1 hour at 37 C, but the reaction can be carried out at room temperature or for longer incubation times. Incubate the Buffer Blank, Positive and Negative Control tubes, and all Sample Tubes at the desired temperature and time. If you are planning to incubate the samples for more than 1 hour, cover the reactions to protect from light. 2. Stop the reaction by adding 25 µl of 60% TCA to each reaction and transfer to 1.0 ml of Protease Assay Buffer (equilibrated to room temperature) in a 10 x 75 mm borosilicate test tube. 3. Gently vortex the tubes to mix and allow the tubes to sit at room temperature. Read after 10 minutes. D. Run the Beacon Protocol 1. Press ENT when the 1) Run Protocol prompt is displayed, type in the desired Protocol ID #, and press ENT. 2. When prompted to Insert Blank, insert the Buffer Blank tube. The fluorescence polarization and intensity of the buffer blank will be subtracted from all subsequent samples. 3. The Beacon 2000 Analyzer is now ready to read the control and sample tubes. Place them into the sample chamber. The LCD display of the Beacon 2000 Analyzer will tell you when a measurement is finished and ready for the next sample. The data are printed on the Beacon printer. After all tubes have been measured, press STOP. 4. Proceed to Section 5.0 for data analysis. E. Endpoint Procedure for High-Background Samples If you suspect that your sample contains a high fluorescence background you must compensate for this background using a Sample Blank containing all components of a sample tube except FSE-casein substrate. Prepare all sample tubes as outlined in the procedure, except prepare them in duplicate. Add substrate to only one tube in each duplicate set. The substrate-free tube will then be used as a Sample Blank. Create a new Beacon 2000 protocol using the same settings as the endpoint assay protocol above except choose the Before Sample blank mode. Using the Before Sample protocol, measure the fluorescence in each sample tube by first measuring the specific blank for that sample. The Beacon 2000 analyzer display will prompt you for each blank and sample. 4.3 Quantitative Protease Assay To more precisely quantitate protease activity, you must first develop a protease standard curve that will be used to determine the range of protease concentrations that can be resolved in your assay. However, unless you are certain of the type of protease activity you are testing you will only be able to report your activity in relative units. The example below is a typical chymotrypsin activity standard curve generated by an endpoint assay. Increasing chymotrypsin concentrations U were added to Incubation Buffer and FSE-casein. A standard endpoint assay was performed with a 1 hour incubation at 37 C. Log protease concentration was plotted vs. polarization (mp). This assay was considered quantitative between and 0.1 units. 160 Chymotrypsin Sensitivity Polarization (mp) Units Chymotrypsin A. Create a Beacon Protocol Please refer to the Beacon 2000 Operators Manual for detailed information regarding the operation of the Beacon 2000 Analyzer. Turn on the analyzer. It will perform a diagnostic procedure and produce a warning message if the RS-232 port or the printer are not online. Press ENT (Enter) to acknowledge any RS-232 or Printer not connected warnings, as these must be brought online before sending data to them. The instrument will then display the first item of the Main Menu 1) Run Protocol. Allow the instrument to warm up for at least 20 minutes before further use.

5 Part # P2010 Lit. # L0054 Rev. 03/01 Page 5 of 7 Please refer to the Beacon 2000 Operator's Manual for detailed information regarding the operation of the Beacon 2000 Analyzer. Turn on the Beacon 2000 Analyzer. The instrument will perform a diagnostic procedure and produce a warning message if the RS-232 port or the printer are not online. Press ENT to acknowledge any RS-232 or Printer not connected warnings, as these devices must be brought online before you can send data to them. The instrument will then display the first item of the Main Menu 1) Run Protocol. Allow the instrument to warm up for at least 20 minutes before further use. 1. If this protocol has been programmed into the Beacon 2000 previously, proceed to step B. Always confirm that the protocol printed on the thermal paper printout is the correct protocol selected. The general procedure for creating a Beacon protocol is as follows: 2. Using the UP and DWN keys, toggle to the Create Protocol option and press the ENT key. The Beacon 2000 will report the number of open protocol numbers. Press ENT again. 3. For the Protocol ID #, type 0 and 3 (or any number between 03 and 99 that has not already been defined). Press ENT. 4. Enter a 4-digit password using the numerical part of the instrument keypad and press ENT. 5. From the Select Read Mode menu use the UP and DWN keys to select Static and then press ENT. From the Select Blank Type menu, select Single Blank, and then press ENT to input a default Blank Delay of 0 seconds. 6. Press ENT to input a default Sample Delay of 0 seconds. 7. At the Number of Average Read Cycles prompt, type 3, then ENT. 8. Press ENT to select the default Set Temperature setting and press 2 and 5 to set the chamber temperature to 25 C. 9. Press ENT two more times to select the default Auto Range and Printer options. You may review a hard copy of the protocol by pressing YES to the Print Protocol prompt. 10. If you have created the protocol correctly, press YES, at the Store New Protocol? prompt and the protocol will be saved under the designated Protocol ID number. B. Prepare Reagents 1. Determine how many reactions are needed. Referring to Section 4.2.B, each set of assays should include one negative, and one Buffer Blank control tubes. Additionally, set up enough reactions for a protease standard curve (typically 5 points for the curve). The standard and sample tubes will contain up to 100 µl of your sample. Single tube reaction recipes are described in Section 4.2.B. Accounting for fluorescence background in your sample may be necessary if the fluorescence background of your sample is greater than 10% of the F-RNA fluorescence intensity. For each sample reaction tube, you will need to include a separate Blank tube that contains only the sample. This modified procedure is described in detail in Section 4.2.E 2. Set up a Master Mix for the negative control in addition to the standard curve and test samples. Refer to the single tube reaction recipe in Section 4.2.B and the example in Section 4.2.B to calculate how the master mix should be made. Aliquot the reaction mix into autoclaved 0.5 ml or 1.5 ml microcentrifuge tubes. C. Perform the Assay 1. Standard reaction conditions are 1 hour at 37 C, but the reaction can be carried out at room temperature or for longer incubation times. Incubate the Buffer Blank, Negative Control, and all Sample Tubes at the desired temperature and time. If you are planning to incubate the samples for more than 1 hour, cover the reactions to protect from light. 2. Stop the reaction by adding 25 µl of 60% TCA to each reaction and transfer to 1.0 ml of Protease Assay Buffer (equilibrated to room temperature) in a 10 x 75 mm borosilicate test tube. 3. Gently vortex the tubes. Read after 10 minutes. D. Run the Beacon Protocol 1. Press ENT when the 1) Run Protocol prompt is displayed, type in the desired Protocol ID #, and press ENT. 2. When prompted to Insert Blank, insert the Buffer Blank tube. The fluorescence polarization and intensity of the buffer blank will be subtracted from all subsequent samples. 3. The Beacon 2000 Analyzer is now ready to read the control and sample tubes. Place them into the sample chamber. The LCD display of the Beacon 2000 Analyzer will tell you when a measurement is finished and ready for the next sample. The data are printed on the Beacon printer. After all tubes have been measured, press STOP. 4. Proceed to Section 5.0 for data analysis. 4.4 Kinetic Assay Suggested Procedure A. Create a Beacon Protocol 1. Using the UP and DWN keys, toggle to the Create Protocol option and press the ENT key. The Beacon will report the quantity of available protocol numbers. Press ENT again. 2. For the Protocol ID #, type 0" and "4 (or any number between 04 and 99 that has not already been defined)and press ENT. Enter a 4-digit password using the numerical part of the instrument keypad and press ENT. 3. From the Select Read Mode menu use the UP and DWN keys to select Kinetic and then press ENT. From the Select Blank Type menu, select YES, then press ENT to input a default Blank Delay of 0 seconds. To the Initial Read prompt press YES,

6 Part # P2010 Lit. # L0054 Rev. 03/01 Page 6 of 7 then press ENT to input a default Sample Delay of 0 seconds. At the Number of Average Read Cycles prompt, type "3", then ENT. 4. From the Read Interval prompt, press ENT twice to set the read interval to 0 minutes and 0 seconds. The read interval will actually be 10 seconds because the Beacon 2000 Analyzer requires a minimum of 4 seconds plus 2 times the read cycle number to produce a measurement (see page 23 of the Beacon 2000 instruction manual for a full description of minimum read time). 5. Press ENT to select the default Set Temperature setting and enter your desired reaction temperature to set the chamber temperature. Press ENT two more times to select the default Auto Range and Printer options. You may review a hard copy of the protocol by pressing YES to the Print Protocol prompt. 6. If you have created the protocol correctly, press YES, at the Store New Protocol? prompt and the protocol will be saved under the designated Protocol ID #. B. Perform the Assay 1. Add 1.0 ml of Incubation Buffer OR your specific activity buffer (see Section 4.2.E to determine if your buffer has high background fluorescence) to a 10 x 75 mm borosilicate tube. 2. Equilibrate the buffer to the assay temperature. 3. Set up the Beacon 2000: a. Press ENT when the 1) Run Protocol prompt is displayed. b. Type in the desired Protocol ID #, and press ENT. c. When prompted to Insert Blank, insert the 10 x 75 mm borosilicate tube containing 1.0 ml of Buffer into the Beacon 2000 sample chamber and close the lid. The fluorescence polarization and intensity of the blank will be subtracted from all subsequent measurements. 4. The Beacon 2000 will then prompt you for an Initial Read. Add 25 µl of FSE-casein to the blank tube and place in the sample chamber and close the lid. The initial read allows you to read the fluorescence polarization before the reaction is started. 5. Add your protease sample to the tube, mix gently, quickly place the tube back in the sample chamber, and close the lid. The Beacon 2000 instrument will keep reading the sample every 6 seconds until you stop the measurement by pressing STOP on the Beacon 2000 keypad. 6. If your protease sample has a high fluorescence background, you may need to subtract the background out using a sample blank. In this case, use a protocol that does not contain an Initial Read. When prompted for the blank, add the protease sample and buffer to a 10 x 75 mm borosilicate tube, place in the sample chamber and close the lid. The fluorescence background of the protease sample in incubation buffer will be subtracted from subsequent polarization measurements. Next, add 25 µl of FSEcasein to the tube to start the reaction and vortex to mix, place the reaction tube back in the sample chamber, and close the lid. 5.0 DATA ANALYSIS The protease activity can be expressed in terms of a change in polarization using the Equation #1: Equation #1: mp = (mp of negative control sample mp) The protease activity can also be expressed in terms of a percentage change of total degradation using Equation #2: Equation #2: % mp = (mp of negative control sample mp) x 100 (mp of negative control mp of positive control) 5.1 Endpoint Assay The mp value of the negative control represents the polarization of the substrate. The mp value of the positive control represents the mp value of the fully degraded product. Therefore, in equation 5.0.2, the denominator represents the maximum mp possible, and the numerator represents the measured mp. Presence of protease is revealed by a reduction in mp as compared to the negative control. Alternatively, the data can be normalized as a percentage of the positive control (100% degradation). For example, if the polarization of the negative control is 140, and the positive control is 40, and the test sample is 70, then: % mp = [(140-70)/(140-40)] x 100 = 70%. Note: The data can be easily copied to spreadsheet or curve-fitting software for analysis on an IBM-compatible PC using the Beacon 2000 Data Management Software. Please refer to the Beacon 2000 Operators Manual for instructions on the use of this software.

7 Part # P2010 Lit. # L0054 Rev. 03/01 Page 7 of Quantitative Assay Construct a standard curve from your raw data using standard linear regression software. Be sure to visualize the curve to make sure you are using only the linear portion of the curve to estimate the activity of your unknown sample. 6.0 COMMONLY ASKED QUESTIONS Is there too much protein in my assay tube? A high concentration of protein (>5 mg) may be hard to follow in a kinetic assay if the protein has some affinity for FSE-casein or FSE-peptides. This would result in high polarization values even if there was protease activity present (e.g., FSE-peptides binding to your sample). In this case, it may be better to use an endpoint assay. The Assay Buffer used in the endpoint assay contains 0.5% SDS and should dissociate any non-specific protein/protein interactions. What if my incubation buffer has a very high background? If possible, use reagents with the lowest possible fluorescence background. Beacon Ultralow Fluorescence Grade Reagents from PanVera are specifically designed for fluorescence applications. If the fluorescence background is still too high in your incubation buffer or protease sample, you can increase the substrate concentration 2- to 4-fold without a significant loss in assay sensitivity. The performance of this product is guaranteed for six months from the date of purchase if stored and handled properly. The Protein Company is a trademark of PanVera Corporation. Beacon and CoreHTS are registered trademarks of PanVera Corporation. Triton is a registered trademark of Union Carbide. Unless specified otherwise, all PanVera products are sold for research use only. 2001, PanVera Corporation. All rights reserved.