| Product Description | Aladdin's UltraBio™ Bacterial Active Protein Extraction Reagent allows for rapid extraction of proteins in E. coli without the need of ultrasonication or high-pressure crushing. The extracted proteins are of high quality, high yield, and high activity. It can be used not only to extract soluble proteins, but also to wash and remove cell debris adhering to the surface of inclusion bodies to obtain high-purity inclusion bodies. It should be noted that this product can not be used to extract proteins from inclusion bodies.This product is optimized to be suitable for BL21 bacteria, DH5ɑ, JM109, and other similar bacteria as well.This product is a buffer solution containing 40mM Tris (pH 8.0) and a specific mild non-denaturing detergent that has been carefully selected to ensure that proteins can be extracted efficiently with activities well preserved. The extracted proteins can maintain their original spatial structures and biological activities, and can be used in a variety of biochemistry and cell biology applications, such as purification of His- or GST-tagged proteins, ELISA, Western Blot, IP, enzyme activity assay, and fluorescent protein assay, etc.This product can quickly and efficiently lyse bacteria, extracting cytosol and nuclear protein through a one-step method (15min) without ultrasonication or high-pressure crushing. Compared with other methods such as ultrasonication, high-pressure crushing and lysozyme treatment, target protein obtained using this product usually have higher yield and activity.The protein concentration of sample lysate obtained using this product can be determined with Aladdin's BCA Protein Assay Kit or the Detergent Compatible Bradford Protein Assay Kit .Please refer to Figure 1 for the performance of this product in lysing BL21 (DE3) expressing GST-EGFP.Figure 1. Lysis of BL21 (DE3) expressing GST-EGFP with Aladdin's UltraBio™ Bacterial Active Protein Extraction Reagent . A. Fluorescence intensity of sample lysate obtained after bacterial lysis for different time courses as indicated. Twenty micrograms of BL21 (DE3) bacteria expressing GST-EGFP were lysed in 1ml of UltraBio™ Bacterial Active Protein Extraction Reagent for different time courses, and the fluorescence of supernatant after centrifugation was measured. As shown in the figure, the bacteria can be fully lysed in 15 minutes. B. SDS-PAGE of supernatants and precipitates. Lanes 1, 3, 5, 7, 9, 11, 13 are supernatants of 1, 2, 5, 10, 15, 20, 30 minutes lysis, respectively, and lanes 2, 4, 6, 8, 10, 12, 14 are their corresponding precipitates. As shown in the figure, after 15 minutes of lysis, the distribution ratio of GST-EGFP in the supernatant and the precipitate is basically unchanged. This figure is for reference only, which may vary due to different experimental conditions.Please refer to Figure 2 for the comparison between UltraBio™ Bacterial Active Protein Extraction Reagent and ultrasonic method in lysing BL21 (DE3) expressing GST-EGFP.Figure 2. Lysis of BL21 (DE3) expressing GST-EGFP by UltraBio™ Bacterial Active Protein Extraction Reagent and ultrasonic method. A. Fluorescence intensity of sample lysate obtained after bacterial lysis for different time courses as indicated. Twenty micrograms of BL21 (DE3) bacteria expressing GST-EGFP were lysed in 1ml of UltraBio™ Bacterial Active Protein Extraction Reagent or lysed in 1ml of PBS by ultrasonication for indicated time courses, and the fluorescence of supernatant after centrifugation was measured. B. SDS-PAGE of supernatants and precipitates. Lanes 1, 3, 5, 7 are supernatants of 5, 10, 15, 20 minutes lysis, respectively, and lanes 2, 4, 6, 8 are their corresponding precipitates. As shown in the figure, the extraction effect of UltraBio™ Bacterial Active Protein Extraction Reagent is significantly better than that of ultrasonic method. This figure is for reference only, which may vary due to different experimental conditions.
Precautions： We recommend adding Protease inhibitor cocktail for bacterial cell extracts (100X) , Protease inhibitor cocktail for purification of His-tagged proteins (100X) or PMSF (100mM) to this product to inhibit protein degradation. We also recommend adding Zonase™ Super Nuclease to this product to reduce the viscosity of the extracted protein.This product is formulated in Tris-HCl buffer. Same buffer system can be used for protein purification after protein extraction.If the extraction effect is not satisfactory for some special strains, freezing and thawing of bacteria can be attempted, which can usually further improve the protein extraction.If the extracted His-tag recombinant protein is going to be purified with nickel column, EDTA should not be used generally for protein extraction, as EDTA will destroy most metal chelation purification resins including the Ni-NTA resin. However, His-tag Purification Resin that is EDTA compatible can be used to purifiy His-tagged proteins from protein extracts containing EDTA.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use： 1. Small-scale extraction of soluble proteins.This extraction method is usually used to determine whether the target protein is expressed.a. Follow conventional methods to cultivate recombinant protein expression strains and induce the expression of target proteins.b. Take 1.5ml of bacterial culture with an OD600 of 0.5-2.0, centrifuge at 12,000-16,000×g at 4℃ or room temperature for 2min, and discard the supernatant.c. Resuspend bacteria with 0.2-0.4ml of Bacterial Active Protein Extraction Reagent by pipetting or vortex. Incubate at room temperature for 15 minutes.d. Centrifuge at 12,000-16,000×g ( better at 16,000×g) at 4℃ for 5min.e. Carefully aspirate the supernatant containing soluble proteins into a new centrifuge tube. Do not touch the pellet when aspirating the supernatant.f. Check the target protein level in the supernatant by SDS-PAGE or Western blot. The recommended loading volume of each sample for SDS-PAGE is 5-15μl.2. Large-scale extraction of soluble proteins.a. Follow conventional methods to cultivate recombinant protein expression strains and induce expression of target proteins.b. Collect 250ml of bacterial culture with an OD600 of about 2.0, centrifuge at 5,000×g at 4℃ or at room temperature for 10 minutes, and discard the supernatant to obtain about 1g of bacteria pellet which can be lysed immediately or frozen at -20℃ to be lysed later. Higher protein yield can be obtained from frozen bacteria, but the freeze-thaw may have a negative effect on the activity of some proteins.c. Add 20-50ml of Bacterial Active Protein Extraction Reagent (appropriate protease inhibitor can be added before use) per gram of bacteria pellet, and resuspend bacteria completely by pipetting. More or less Protein Extraction Reagent can be used to reduce or increase the protein concentration in the crude extract. If it is easier to obtain more wet bacteria, it is recommended to use less extraction reagents. Additionally, add lysozyme ( Super Nuclease can also be added to reduce the viscosity of the crude extract.d. Incubate at room temperature for 15 minutes.e. Centrifuge at 12,000-16,000×g (better at 16,000×g) for 10 minutes at 4℃ or room temperature.f. Carefully aspirate the supernatant containing soluble proteins into a new centrifuge tube. Do not touch the pellet when aspirating the supernatant.g. Check the target protein level in the supernatant by SDS-PAGE or Western blot. The recommended loading volume of each sample for SDS-PAGE is 5-15μll.FAQ:1. The yield of target protein is low.a. Insufficient bacterial lysis. Repeated freeze-thaw of bacteria or the addition of lysozyme can improve the protein extraction.b. The viscosity of the sample is too high. The addition of Super Nuclease or DNase I can reduce the viscosity of the crude extract and facilitate the extraction of soluble proteins.c. Target protein is degraded. Add protease inhibitors in Protein Extraction Reagent can reduce the degradation of target proteins. We recommend using Protease Inhibitor Cocktail for Bacterial Cell Extracts (100X) , Protease Inhibitor Cocktail for Purification of His-tagged Proteins (100X) or PMSF (100mM) produced by Super Nuclease ( Super Nuclease, ≥99%100KUD7121-2000KU Super Nuclease, ≥99 00KUD7121-25KU Super Nuclease, ≥99%25KUD7121-500KU Super Nuclease, ≥99%500KUD7121-5KU Super Nuclease, ≥99%5KUD7126-100KU Super Nuclease, ≥99%, with His-tag100KUD7126-2000KU Super Nuclease, ≥99%, with His-tag2000KUD7126-25KU Super Nuclease, ≥99%, with His-tag25KUD7126-500KU Super Nuclease, ≥99%, with His-tag500KUD7126-5KU Super Nuclease, ≥99%, with His-tag5KUP0006CDetergent Compatible Bradford Protein Assay Kit800P0009Enhanced BCA Protein Assay Kit5000TP0010Enhanced BCA Protein Assay Kit500TP0010SEnhanced BCA Protein Assay Kit200TP0011BCA Protein Assay Kit5000TP0012BCA Protein Assay Kit500TP0012SBCA Protein Assay Kit200TP0013Cell Lysis Buffer for Western or IP100mlP0013BRIPA Lysis Buffer 100mlP0013CRIPA Lysis Buffer (Medium)100mlP0013DRIPA Lysis Buffer (Weak)100mlP0013ERIPA Lysis Buffer Set 50ml eachP0013FNP-40 Lysis Buffer100mlP0013GSDS Lysis Buffer100mlP0013JCell lysis buffer for Western and IP without inhibitors100mlP0013KRIPA lysis buffer 100mlP0013M-100ml Mammalian Active Protein Extraction Reagent100mlP0013Q-100ml Bacterial Active Protein Extraction Reagent100mlP0013Q-500ml Bacterial Active Protein Extraction Reagent500mlP0013S-100ml Insect Active Protein Extraction Reagent100mlP0027Nuclear and Cytoplasmic Protein Extraction Kit50TP0028Nuclear and Cytoplasmic Protein Extraction Kit100TP0033Membrane and Cytosol Protein Extraction Kit100TP1005Protease inhibitor cocktail for general use, 100X1mlP1006Protease inhibitor cocktail for general use, 100X5mlP1008Protease inhibitor cocktail for general use, MS-SAFE, 50X1mlP1009Protease inhibitor cocktail for general use, MS-SAFE, 50X5mlP1010Protease inhibitor cocktail for mammalian cell and tissue extracts, 100X1mlP1011Protease inhibitor cocktail for mammalian cell and tissue extracts, 100X5mlP1015Protease inhibitor cocktail for plant cell and tissue extracts, 100X1mlP1016Protease inhibitor cocktail for plant cell and tissue extracts, 100X5mlP1020Protease inhibitor cocktail for fungal and yeast extracts, 100X1mlP1021Protease inhibitor cocktail for fungal and yeast extracts, 100X5mlP1025Protease inhibitor cocktail for bacterial cell extracts, 100X1mlP1026Protease inhibitor cocktail for bacterial cell extracts, 100X5mlP1030Protease inhibitor cocktail for purification of His-tagged proteins, 100X1mlP1031Protease inhibitor cocktail for purification of His-tagged proteins, 100X5mlP1045Protease and phosphatase inhibitor cocktail for general use , 50X2mlP1046Protease and phosphatase inhibitor cocktail for general use, 50X10mlP1048Protease and phosphatase inhibitor cocktail for general use, MS-safe, 50X1mlP1049Protease and phosphatase inhibitor cocktail for general use, MS-safe, 50X5mlP1050Protease and phosphatase inhibitor cocktail for mammalian cell and tissue extracts, 50X2mlP1051Protease and phosphatase inhibitor cocktail for mammalian cell and tissue extracts, 50X10mlP1055Protease and phosphatase inhibitor cocktail for plant cell and tissue extracts, 50X2mlP1056Protease and phosphatase inhibitor cocktail for plant cell and tissue extracts, 50X10mlP1060Protease and phosphatase inhibitor cocktail for fungal and yeast extracts, 50X2mlP1061Protease and phosphatase inhibitor cocktail for fungal and yeast extracts, 50X10mlP1065Protease and phosphatase inhibitor cocktail for bacterial cell extracts, 50X2mlP1066Protease and phosphatase inhibitor cocktail for bacterial cell extracts, 50X10mlP2210 His-tag Purification Resin (耐还原螯合型)10mlP2210FTHis-tag Purification Resin2mlP2220 His-tag Purification Resin1000mlP2226His-tag Protein Purification Kit10mlS3090Cell and Tissue Lysis Buffer for Nitric Oxide Assay100mlST206Lysozyme (＞20KU/mg)0.5gST506PMSF(100mM)10ml
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