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Β-Lactamase Activity Assay Kit

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Β-Lactamase Activity Assay Kit Technical Bulletin β-Lactamase Activity Assay Kit Catalog Number MAK221 Product Description Positive Control 1 vial β-Lactamase (βL; EC 3.5.2.6) is an enzyme Catalog Number MAK221C first identified in Escherichia coli and has L Hydrolysis Buffer 100 µL been described as penicillinase. A number of Catalog Number MAK221D βLs have since been identified from various bacteria. βLs specifically hydrolyze β-lactam Reagents and Equipment rings present in antibiotics such as penicillin, Required but Not Provided cephalosporins, monobactam, and Clear flat-bottom 96-well plates. Cell carbapenem, and confer resistance against culture or tissue culture treated plates these antibiotics.1,2 are not recommended. The β-Lactamase Activity Assay Kit provides Spectrophotometric multiwell plate a simple assay for measuring βL activity in reader βL-secreting bacteria in fermentation media DMSO (dimethyl sulfoxide) (Catalog and bacterial cultures. It can also be used to Number D2650 or equivalent) detect βL-secreting bacteria in saliva, urine, and serum of infected mammals and in food Precautions and Disclaimer samples. βL activity is measured by For R&D use only. Not for drug, household, hydrolyzing a chromogenic cephalosporin or other uses. Please consult the Safety called nitrocefin, producing a colorimetric Data Sheet for information regarding hazards product with an absorbance maximum at and safe handling practices. 490 nm proportional to the enzymatic activity present. One unit of β-lactamase is Preparation Instructions the amount of enzyme required to hydrolyze Briefly centrifuge vials before opening. Use 1.0 µmole of nitrocefin per minute at pH 7.0 ultrapure water for the preparation of at 25 C. reagents. To maintain reagent integrity, avoid repeated freeze/thaw cycles. Components β-Lactamase Assay Buffer and βL Hydrolysis The kit is sufficient for 100 assays in 96-well Buffer: Allow buffers to come to room plates. temperature before use. -Lactamase Assay Buffer 27 mL Nitrocefin (in DMSO): Store at –20 C, Catalog Number MAK221A protected from light. Warm to room Nitrocefin, in DMSO 220 µL temperature before use to melt DMSO. Catalog Number MAK221B Use within 2 months. Page 1 of 6 Positive Control: Reconstitute with 20 µL of Add 1–50 µL of the samples into duplicate β-Lactamase Assay Buffer. Mix well by wells. Bring samples to a final volume of pipetting. Aliquot and store at –20 C. 50 µL using β-Lactamase Assay Buffer. Use within 2 months. Notes: For unknown samples, it is suggested Storage/Stability to test several sample dilutions to ensure the The kit is shipped on wet ice and storage at readings are within the linear range of the –20 C, protected from light, is standard curve. recommended. For samples exhibiting significant background, include a Sample Blank for each Procedure sample by omitting the Nitrocefin. The All samples and standards should be run in Sample Blank readings can then be duplicate. subtracted from the sample readings. Nitrocefin Standards For a positive control (optional), dilute 2 µL Hydrolyze the Nitrocefin stock solution by of the Positive Control solution with 8 µL of mixing 4 µL of Nitrocefin, 8 µL of L β-Lactamase Assay Buffer and mix well. Add Hydrolysis Buffer, and 28 µL of DMSO to 1–10 µL of the diluted Positive Control prepare a 2 mM (2 nmole/µL) Nitrocefin solution to the desired wells. Adjust the final Standard Solution. Incubate at 60 C for volume to 50 µL using β-Lactamase Assay 30 minutes. Cool to room temperature and Buffer. centrifuge. Prepare hydrolyzed Nitrocefin Assay Reaction Standard Solution fresh, just prior to each use. 1. Set up a Reaction Mix according to the scheme in Table 1. 50 µL of Reaction Mix Add 0, 2, 4, 6, 8, and 10 µL of the is required for each reaction (well). hydrolyzed 2 mM (2 nmole/µL) Nitrocefin Standard Solution into wells of a 96-well Table 1. plate generating 0 (blank), 4, 8, 12, 16, and Reaction Mix 20 nmole/well standards. Add -Lactamase Assay Buffer to each well to bring the total Reagent Volume volume to 100 µL. -Lactamase 48 µL Sample Preparation Assay Buffer Clear liquid samples can be assayed directly. Nitrocefin 2 µL For bacterial samples, centrifuge the samples at 10,000 g for 10 minutes in a pre- 2. Add 50 µL of the Reaction Mix to wells weighed centrifuge tube. Discard the containing samples and Positive Control. supernatant and determine the weight of the Mix well using a horizontal shaker or by pellet. Resuspend the pellet with 5 µL of pipetting. β-Lactamase Assay Buffer per mg of sample. 3. Measure the absorbance at 490 nm Sonicate samples for 5 minutes and place (A490) in a microplate reader in kinetic them on ice for 5 minutes. Centrifuge the mode for 30-60 minutes at room samples at 16,000 g at 4 C for temperature. Protect the plate from light 20 minutes. Transfer supernatant to a fresh during the incubation. It is recommended tube. to take absorbance readings every minute. Page 2 of 6 Note: Incubation time depends on the β-Lactamase Activity activity of β-Lactamase in the samples. β-Lactamase activity = Sa 4. Continue taking measurements until the (Reaction Time) Sv value of the most active sample is greater than the value of the highest standard (20 nmole/well). At this time where: the most active sample is near or Sa = Amount of Nitrocefin (nmole) exceeds the end of the linear range of hydrolyzed in unknown sample well the standard curve. between T1 and T2 from standard Note: The Nitrocefin Standards can be read curve at the end of the incubation time. Reaction Time = T2 – T1 (minutes) Results Sv = Sample volume (mL) added to well Calculations Plot the absorbance (A490) for each well -Lactamase activity is reported as versus time. nmole/min/mL = milliunit/mL. Correct for the background by subtracting One unit of -lactamase is the amount of the measurement obtained for the 0 (blank) enzyme required to hydrolyze 1.0 µmole of Nitrocefin standard from that of the nitrocefin per minute at pH 7.0 at 25 C. standards, controls, and samples. Background values can be significant and must be subtracted from all readings. Use the values obtained from the appropriate Sample Calculation: Nitrocefin Standards to plot a standard Amount of Nitrocefin (S ) = 5.84 nmole curve. a (from standard curve) Note: A new standard curve must be set up (T1) = 3 minutes each time the assay is run. (T2) = 32 minutes Choose two time points (T1 and T2) in the linear range of the plot and determine the Sample volume (Sv) = 0.050 mL A490 at each time (ABS1 and ABS2). Note: It is essential that ABS1 and ABS2 fall within the linear range of the standard curve. β-Lactamase activity in sample well: Calculate the change in absorbance from T1 nmole/min/mL (milliunits/mL) = to T2 for the samples. 5.84 nmole/well = ABS = ABS2 – ABS1 (32 min – 3 min) 0.050 mL /well Subtract the Sample Blank ABS value from 4.03 milliunits/mL the Sample ABS reading to obtain the corrected measurement. Using the corrected measurement, determine the amount of Nitrocefin (nmole/well) generated by the β-Lactamase assay between T1 and T2 (Sa). Page 3 of 6 References 2. Drawz, S.M., and Bonomo, R.A., Three 1. Abraham, E.P., and Chain, E., An enzyme decades of beta-lactamase inhibitors. from bacteria able to destroy penicillin. Clin. Microbiol. Rev., 23, 160–201 Nature, 146, 837 (1940). (2010). Troubleshooting Guide Problem Possible Cause Suggested Solution Assay Buffer must be at room Cold assay buffer temperature Refer and follow Technical Bulletin Assay not Omission of step in procedure working precisely Plate reader at incorrect Check filter settings of instrument wavelength Type of 96-well plate used For colorimetric assays, use clear plates Samples prepared in different Use the Assay Buffer provided or refer buffer to Technical Bulletin for instructions Repeat the sample homogenization, Cell/Tissue culture samples were increasing the length and extent of incompletely homogenized Samples with homogenization step. erratic Samples used after multiple Aliquot and freeze samples if needed to readings freeze-thaw cycles use multiple times Presence of interfering substance If possible, dilute sample further in the sample Use of old or inappropriately Use fresh samples and store correctly stored samples until use Thaw all components completely and Improperly thawed components mix gently before use Use of improperly stored reagents Store the components appropriately Lower/higher readings in Allowing the reagents to sit for Prepare fresh Reaction Mixes before samples and extended times on ice each use Refer to Technical Bulletin and verify standards Incorrect incubation times or correct incubation times and temperatures temperatures Use calibrated pipettes and aliquot Incorrect volumes used correctly Use of partially thawed Thaw and resuspend all components Non-linear components before preparing the Reaction Mixes Pipetting errors in preparation of standard Avoid pipetting small volumes curve standards Pipetting errors in the Reaction Prepare Reaction Mixes whenever Mix possible Page 4 of 6 Problem Possible Cause Suggested Solution Pipette gently against the wall of the Air bubbles formed in well plate well Non-linear Standard stock is at incorrect Refer to the standard dilution standard concentration instructions in the Technical Bulletin curve Recheck calculations after referring to Calculation errors (continued) Technical Bulletin Substituting reagents from older Use fresh components from the same kits/lots kit Samples measured at incorrect Check the equipment and filter settings wavelength Samples contain interfering Unanticipated If possible, dilute sample further results substances Sample readings above/below the Concentrate or dilute samples so linear range readings are in the linear range Page 5 of 6 Notice We provide information and advice to our customers on application technologies and regulatory matters to the best of our knowledge and ability, but without obligation or liability.
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