| Description | Firefly Luciferase Reporter Gene Assay Kit (Glow) is a glow-type quantitative detection kit characterized by high sensitivity and stable luminescent signals. It is suitable for high-throughput detection of firefly luciferase expression in mammalian cells. This product significantly enhances Firefly Luciferase Reporter Gene Assay Kit (Glow) is a glow-type quantitative detection kit characterized by high sensitivity and stable luminescent signals. It is suitable for high-throughput detection of firefly luciferase expression in mammalian cells. This product significantly enhances luminescent signal intensity, effectively improving detection sensitivity. It can be used to detect firefly luciferase reporter gene expression in ADCC Reporter Bioassays or cells. G1375755Component10 mL100 mLStorageG1375755AFirefly Luciferase Reaction Buffer (Glow)10 mL100 mL-20℃. Store in the dark.G1375755BFirefly Luciferase Substrate (Glow)1 vial1 vial-20℃. Store in the dark.he main features and differences among three One-Step Firefly Luciferase Reporter Gene Assay Kits from Aladdin are as follows:For high luminescent signal requirements and detection within 30 minutes: Recommended: Firefly Luciferase Reporter Gene Assay Kit (Glow Bright) (Cat. No. B1375760)For both luminescent signal and stability requirements, suitable for batch or continuous operation: Recommended: Firefly Luciferase Reporter Gene Assay Kit (Cat. No. F773892)For standard luminescent signal requirements: Recommended: Firefly Luciferase Reporter Gene Assay Kit (Glow) (Cat. No. G1375755)For routine laboratory detection, the Firefly Luciferase Reporter Gene Assay Kit (Glow Bright) (Cat. No. B1375760) is the recommended priority. Product Cat. No.Product NameLuminescence TypeSignal IntensitySensitivityHalf-LifePackagingSuitable for Batch SamplesOptimal Use After Mixing & Storing at -20°CF773892Firefly Luciferase Reporter Gene Assay KitGlowRelatively HighRelatively SensitiveUp to 2h1 bottle Buffer1 vial SubstrateVery Suitable1 MonthG1375755Firefly Luciferase Reporter Gene Assay Kit (Glow)GlowHighSensitive25-30min1 bottle Buffer1 vial SubstrateVery Suitable1 WeekB1375760Firefly Luciferase Reporter Gene Assay Kit (Glow Bright)GlowVery HighHighly Sensitive25-30min1 bottle Buffer1 vial SubstrateVery Suitable1 WeekInstructions for Use:1. User-Supplied MaterialsPBSMultichannel pipetteWhite or black opaque cell culture microplateLuminometer or microplate reader equipped with a luminescence detection module.2. Preparation Before Assay(1) Initial Reconstitution: Upon first use, transfer the entire contents of the Firefly Luciferase Reaction Buffer (Glow) bottle into the Firefly Luciferase Substrate (Glow) vial. Mix thoroughly until the substrate is completely dissolved. Aliquot the reconstituted detection reagent as needed. For long-term storage, aliquot and store at -70°C. For short-term storage, store aliquots at -20°C for no longer than one month. Use reconstituted reagent promptly.(2) Pre-assay: Before each experiment, equilibrate the aliquoted, frozen detection reagent to room temperature.3. Procedure(1) Equilibrate Plates: Remove the cell culture plate from the incubator and allow it to stand at room temperature for 5-15 minutes.Note: Use white or black opaque cell culture plates to minimize signal interference between wells.(2) Add Detection Reagent: Using a multichannel pipette, add the equilibrated Firefly Luciferase Reaction Buffer (Glow) containing the substrate to each cell culture well. The volume added should be equal to the volume of the culture medium in the well. For example:* 96-well plate: Typically add 80-100 µL culture medium; add 80-100 µL detection reagent.* 384-well plate: Typically add 20-30 µL culture medium; add 20-30 µL detection reagent.(3) Mix to Lyse Cells: To ensure complete cell lysis, place the plate on an orbital shaker or an instrument with shaking capability. Shake at medium to high speed at room temperature for 5 minutes.Note: A shaking time of 5 minutes is recommended. This can be adjusted based on cell density to ensure complete lysis and stable luminescent results.(4) Detection: Following the shaking step, immediately measure firefly luciferase reporter gene activity using a luminometer or microplate reader.Note: For optimal results, perform the detection as soon as possible after adding the detection reagent.PrecautionsInstrument Selection: Any instrument capable of detecting chemiluminescence is suitable for use with this kit. However, background signal levels and measured values for identical samples may vary between different detectors. Values obtained from different instruments for the same sample are not directly comparable. To prevent well-to-well interference, the use of opaque white or black cell culture plates is strongly recommended.Consistent Conditions: Luminescent signals can be influenced by environmental factors such as medium components and temperature. Ensure consistent detection conditions across all samples within an experiment.Temperature Sensitivity: The enzymatic reaction is temperature-sensitive. It is essential to equilibrate both the detection reagent and the cell culture plate to room temperature before adding the reagent and performing the assay.Reagent Stability: To maintain the stability of the luciferase assay reagent, aliquot it appropriately after reconstitution. For long-term storage, store aliquots at -70°C protected from light. For short-term storage, store at -20°C for no longer than one month. Use aliquots promptly and avoid repeated freeze-thaw cycles.Multiple Plate Assay: If detecting multiple cell culture plates simultaneously, strive to maintain consistent incubation time after adding the detection reagent to each plate before reading. This ensures optimal and comparable results across plates.Emission Wavelength: The bioluminescence catalyzed by firefly luciferase exhibits a peak emission wavelength of 560 nm... Read More | Product introduction：Dualucif The firefly & Renilla assay kit (dual luciferase reporter assay kit) provides an effective means to detect the expression of genes. In DLR detection, the activities of firefly luciferase and Renilla luciferase can be detected in a single sample in turn. FirstProduct introduction：Dualucif The firefly & Renilla assay kit (dual luciferase reporter assay kit) provides an effective means to detect the expression of genes. In DLR detection, the activities of firefly luciferase and Renilla luciferase can be detected in a single sample in turn. First, luciferin was used as substrate to detect the activity of firefly luciferase, then substances inhibiting the catalysis of firefly luciferase were added, and coelenterazine was added to detect the activity of Renilla luciferase to achieve dual luciferase reporter gene detection. The bioluminescence system of luciferase and its substrate can detect gene expression very sensitively and efficiently. Usually, the transcriptional regulatory element or 5'promoter region of the gene of interest is cloned upstream of luciferase, or the 3'-utr region is cloned downstream of luciferase to construct a reporter gene plasmid, and then transfect the cells. After the cells are treated with appropriate drugs, the cells are lysed, and the transcriptional regulation effect of drug treatment on the target gene is judged by detecting the luciferase activity. Renilla luciferase is more often used as an internal reference for detecting transfection efficiency to eliminate the difference in cell number and transfection efficiency. Firefly luciferase is a protein with a molecular weight of about 61 kDa. In the presence of ATP, magnesium ions and oxygen, it can catalyze the production of oxyluciferin from luciferin. In the process of luciferin oxidation, it will produce a light signal. Renilla luciferase is a protein with a molecular weight of about 36 kDa. In the presence of oxygen, it can catalyze the oxidation of coelenteramide to coelenteramide, and also produce light signals in the process of coelenteramide oxidation. The optical signal of this kit can be measured by chemiluminescence instrument, microplate reader or liquid scintillation tester. The kit has the characteristics of rapid detection, high sensitivity, wide detection range and no interference of cell endogenous activity.Instruction：1.Cell lysis ( 1 ) Remove the medium and gently wash twice with PBS ( adherent cells can be operated directly, suspension cells need to be centrifuged to collect cells ). Add 1 × Lysis Buffer ( diluted component A with sterile water at 4 : 1 ) according to the following scheme, and then place the culture plate on a micro-oscillator at room temperature for 15 min to fully lyse the cells. Note : The pyrolysis products can be stored at room temperature for 6 h, and can be stored at − 70 °C for a long time ( the pyrolysis products cannot be repeatedly frozen and thawed ). ( 2 ) The pyrolysis products were centrifuged at 10000-15000 rpm for 3-5 min. After centrifugation, the supernatant was transferred into a new EP tube for subsequent detection. Note : Cells can be detected immediately after lysis, or frozen, and re-detected when needed. The frozen samples need to be thawed to room temperature for detection. 2. Preparation of working fluid ( 1 ) Restore all components to room temperature. ( 2 ) Dilute component C with component B to 0.2 mg / mL firefly luciferase working solution. Note : The firefly luciferase working solution cannot be repeatedly frozen and thawed. If the amount of a single experiment is small, it is recommended to be subpackaged into small specifications according to a single amount of use. ( 3 ) The E component was diluted into the renilla luciferase working solution with the D component, and the dilution method was 1 µL E component was added to the 49 µL D component. Note : Renilla luciferase working solution needs to be prepared now. 3.chemiluminescence value detection ( 1 ) According to the operation instructions of the instrument, the instrument with chemiluminescence detection function was opened, such as multifunctional microplate reader. The parameters were set, the determination time was 10 s, and the determination interval was 2 s. ( 2 ) each sample determination, take the sample 20-100 µL ( if the sample volume is enough, please add 100 µL ; if the sample amount is insufficient, the amount can be appropriately reduced, but the amount of detection holes needs to be consistent ). 1 × Lysis Buffer was blank control. ( 3 ) 100 µL firefly luciferase working solution was added to determine the RLU ( relative light unit ) value ( it is recommended that the microplate reader set up the Shaking mixing function ). Note : Since the luminescence is instantaneous, it is recommended to detect immediately after adding the firefly luciferase working solution. ( 4 ) 100 µL renilla luciferase working solution was added to determine the RLU ( relative light unit ) value ( Shaking mixing function is recommended for microplate reader ). ( 5 ) In the case of renilla luciferase as an internal reference, the RLU value measured by firefly luciferase was divided by the RLU value measured by renilla luciferase. According to the obtained ratio, the activation degree of the target reporter gene between different samples was compared. If firefly luciferase is used as an internal reference, similar calculations can also be performed.Component：Recommendation：It is recommended to use component B in advance to prepare 2 mg / mL storage solution, component B, component D and component C prepared as storage solution, and to carry out small batch packing according to the experimental requirements. All test working fluids are recommended to be used now to avoid repeated freezing and thawing.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. in order to obtain the best determination effect, when using a single tube chemiluminescence instrument for determination, the time from the mixing of sample and determination reagent to the pre determination should be controlled as much as possible; When using a multi-functional fluorescent microplate reader with chemiluminescence detection function, it is advisable to add all samples first, and then uniformly add firefly luciferase detection reagent. 3. the strongest wavelength of firefly luciferase catalyzed bioluminescence is 560 nm, and the strongest wavelength of Renilla luciferase catalyzed bioluminescence is 480 nm. 4. to prevent interference between holes, it is recommended to use white opaque orifice plate. 5. due to the influence of temperature on enzyme reaction, the sample and reagent should be measured after reaching room temperature. 6. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Study on gene expression regulation and promoter... Read More | This product can rapidly, gently, and efficiently lyse mammalian cells, effectively extracting cytoplasmic and nuclear proteins. This reagent uses a mild formula to ensure that the extracted protein maintains biological activity and can be applied to various protein analysis experiments, such as This product can rapidly, gently, and efficiently lyse mammalian cells, effectively extracting cytoplasmic and nuclear proteins. This reagent uses a mild formula to ensure that the extracted protein maintains biological activity and can be applied to various protein analysis experiments, such as reporter gene and enzyme activity determination, immune detection, protein purification, etc. The extracted protein can be quantitatively analyzed using the BCA method. The reagent kit contains a mixture of protease inhibitors, which can effectively prevent protein degradation during the protein extraction process.M665813Component100 TStorageM665813AMammalian Protein Extraction Reagent100 mLRTM665813BProtease Inhibitor Cocktail (100×)1 mL-20℃. Avoid freeze/thaw cycle. precautions1. This product can effectively lyse adherent cells cultured on cell culture plates (without scraping) and suspended cells collected by centrifugation, with higher extraction efficiency than repeated freeze-thaw or ultrasound methods. But for the extraction of tissue proteins, it is recommended to use the tissue protein extraction kit (CW0891).The optimal dosage for protein extraction from adherent cells is listed in Table 1. Collecting cells first can reduce the amount of reagents used to obtain higher protein concentrations.3. The amount of extraction reagents used can also be estimated based on the number of cells. If 2 × 106 Hela cells weigh about 20 mg, 200 need to be added µ Extract reagents.4. The protein extracted from this product can be quantitatively analyzed using the BCA method.Operation steps● Protein extraction from adherent cells1. Please remove the required Mammalian Protein Extraction Agent for pre cooling before protein extraction.2. Carefully pour out the culture medium of adherent cells and rinse the cells with PBS.3. Add an appropriate amount of Mammalian Protein Extraction Reagent (add Protein Inhibitor Cocktail in a 1:99 ratio 2-3 minutes before protein extraction), blow adherent cells on ice with a gun tip, transfer the lysate to a centrifuge tube, incubate on ice for 20 minutes, and allow the cells to fully lyse (please refer to Appendix 1 for the amount of reagent used, and the time for placing on ice should be adjusted according to different cell types). 4. Centrifuge at 14000 × g for 5-10 minutes.5. Transfer the supernatant to a new tube for further analysis. ● Suspension cell protein extraction1. Please remove the required Mammalian Protein Extraction Agent for pre cooling before protein extraction.2. Suspend 2500 × g of cells, centrifuge for 10 minutes, and discard the supernatant. Rinse cells with PBS. 2500 × g, centrifuge for 10 minutes, discard the supernatant.3. Add an appropriate amount of Mammalian Protein Extraction Agent, and 2-3 minutes before protein extraction, add Protein Inhibitor Cocktail in a ratio of 1:99, which is 1 x working solution.4. Add at least 1 ml of 1x working solution to every 100 mg of cells. If the extracted sample size is large, a small amount of 1x working solution can be used to resuspend the cells first, and then the remaining working solution can be added.5. After blowing evenly, place it on ice for 20 minutes to allow the cells to fully lyse (the time for placing it on ice should be adjusted according to different cell types). 6. Centrifuge at 14000 × g for 15 minutes.7. Transfer the supernatant to a new tube for further analysis.Table 1. Recommended usage of extraction reagents Cell culture plate type or dish type Extraction reagent usage 100 mm 500-1,000 µl 60 mm 250-500 µl 6-well culture plate 200-400 µl /well 24-well culture plate 100-200 µl /well 96-well culture plate 50-100 µl /well Table 2. Common Problems and Solutions Problem Possible reasons Resolvent Low extraction rate Low protein expression level Optimize transfection system Low extraction rate Insufficient reagent usage Increase the usage of extraction reagents Low extraction rate Reagent unable to dissolve cell membrane Increase cracking time or increase shaking amplitude Unable to obtain membrane protein This product is more suitable for extracting nuclear plasma protein Using eukaryotic cell membrane protein extraction kit... Read More | DescriptionGreen LED Array for Photo KitAlysis high-throughput screening platform. For use with Photo KitAlysis Starter Kit (Z742612). User guide is provided in the below hyperlink.Photo KitAlysis Operating InstructionsFeatures:Designed and tested by synthetic chemists.Controller provides repeatableDescriptionGreen LED Array for Photo KitAlysis high-throughput screening platform. For use with Photo KitAlysis Starter Kit (Z742612). User guide is provided in the below hyperlink.Photo KitAlysis Operating InstructionsFeatures:Designed and tested by synthetic chemists.Controller provides repeatable milliamp selection for photon intensity (sold seperately)527 nm wavelength0-30 mA variable LED outputNon-magnetic LED baseChemically resistant LED coverPTFE coated cablingPhoto Kitalysis Starter Kitrequired for operation (sold separately). Best when used withKitAlysis Benchtop Inertion Box(sold separately)... Read More | This plant protein extraction kit can extract soluble plant proteins from fresh, frozen, or dried plant tissues. Suitable for protein extraction from various plants and different parts of plants (such as roots, stems, leaves, flowers, seeds, etc.), with high extraction efficiency, high protein yieldThis plant protein extraction kit can extract soluble plant proteins from fresh, frozen, or dried plant tissues. Suitable for protein extraction from various plants and different parts of plants (such as roots, stems, leaves, flowers, seeds, etc.), with high extraction efficiency, high protein yield, high activity, and fast speed. The extracted protein can be directly subjected to protein electrophoresis analysis, immunoprecipitation, Western Blot, protein activity determination, and protein purification experiments. The concentration of the extracted protein can be determined using the BCA protein quantification kit. P665757Component100 TStorageP665757APlant Protein Extraction Reagent100 mLRTP665757BProtease Inhibitor Cocktail (100×)1 mL-20℃. Avoid freeze/thaw cycle. Precautions：1. This product contains 1mM EDTA.2. To prevent protein degradation, all operations should be carried out on ice as much as possible.3. After extracting protein using this product, the BCA method can be used for protein quantification.4. To achieve the best experimental results, please adjust the optimal usage amount according to the experiment.Operation steps:1. Please remove the required Plant Protein Extraction Agent for pre cooling before protein extraction.2. Weigh the weight of the experimental plant tissue. Add 5 ml of Plant Protein Extraction Agent to 1 g of tissue (add Protein Inhibitor Cocktail in a 1:99 ratio before protein extraction).Attention:1) Before homogenization, cut large pieces of plant tissue into small pieces and homogenize them with a mechanical homogenizer for 10 seconds, with an interval of 10 seconds. Repeat the process three times and select the appropriate homogenization method according to the different tissue samples.2) The amount of lysate used is adjusted according to different parts of the plant. If concentrated protein extracts are needed, the amount of Plant Protein Extraction Agent used can be appropriately reduced.3. After homogenization, incubate on ice for 20-30 minutes.4.4 ℃ 13400 × g, centrifuge for 20 minutes.5. Collect soluble proteins from the supernatant for further purification or downstream analysis... Read More |