ProteoSpin™ Inclusion Body Protein Isolation Kits For the rapid isolation of inclusion body proteins For research use only and NOT intended for in vitro diagnostics. Powered by Bioz ProteoSpin™ Inclusion Body Protein Isolation Kits For the rapid isolation of inclusion body proteins Price USD$335.00 SKU 10300 Format Micro Maxi Size 25 Preps Quantity Overview Details Documentation Citations Overview All-in-one solution for inclusion body protein isolation and purification Fast and convenient spin column protocol Complete kit with Cell Lysis Reagent, Inclusion Body Solubilization Reagent, buffers and spin columns to purify proteins Purification is based on spin column chromatography that uses Norgen’s resin separation matrix These kits provide everything required to isolate and purify inclusion body proteins from induced bacterial cultures. First a proprietary Cell Lysis Reagent is used to selectively lyse the cells and release inclusion bodies in their solid form. Next, inclusion bodies are dissolved and their contents released using the provided IB Solubilization Reagent. Inclusion body proteins are then further purified using spin columns for rapid and convenient buffer exchange and desalting. This kit provides a convenient way to screen recombinants prior to scaling up. ProteoSpin™ Inclusion Body Protein Isolation Micro Kit The process is efficient and streamlined and can process up to 12 samples in only 60 minutes. Each spin column is able to recover up to 50 µg of acidic or basic proteins. Purified recombinant proteins are then ready for SDS-PAGE, 2D gels, Western blots, Mass Spectrometry analysis, and other applications. ProteoSpin™ Inclusion Body Protein Isolation Maxi Kit The procedure is efficient and streamlined and can process up to 4 samples in approximately 2 hours. Each spin column is able to recover up to 12 mg of acidic or basic proteins from 100 mL of induced bacterial culture. Purified recombinant proteins are then ready for SDS-PAGE, 2D gels, Western blots, Mass Spectrometry analysis, and other applications. About Inclusion Bodies Bacteria are widely used for the expression of different proteins. However, 70-80% of the proteins expressed in bacteria by recombinant techniques are typically contained in insoluble inclusion bodies (i.e., protein aggregates). The protein of interest found in these subcellular structures is often inactive, due to incorrect folding. The production rate of recombinant proteins stored in inclusion bodies is invariably higher than those synthesized as soluble proteins. The reason behind this is thought to be the resistance of insoluble proteins to proteolysis by cellular enzymes. In addition, separation of insoluble recombinant proteins in inclusion bodies is considerably easier than that of soluble proteins. These factors have been the major influences favoring scale-up of high-value proteins using bacterial fermentation for example. Procedures for the purification of the expressed proteins from inclusion bodies are often labour-intensive, time-consuming and not cost-effective. This kit provides the essential reagents for cell disruption, inclusion body solubilization and purification using spin column chromatography - all optimized to work together thereby simplifying the process and saving a tremendous amount of time and cost. Details Supporting Data Previous Figure 1. Isolation of Acidic Recombinant Protein. Small (1.5 mL) cultures of BL21 (DE3) pLysS bacteria expressing a 50 kDa chimeric protein (acidic) were pelleted. Cells were lysed and inclusion bodies separated and dissolved using the ProteoSpinTM Inclusion Body Protein Isolation Micro Kit. The resulting protein was bound to the ProteoSpinTM column, washed and eluted in 50 µL of the provided elution buffer. The eluted protein samples were analyzed in 12.5% polyacrylamide gels, which were run for 45 minutes at 200 V/6.5 cm. The protein bands were made visible by staining with Coomassie Blue R-250. Figure 2. Isolation of Basic Recombinant Protein (RNase A). Small (1.5 mL) cultures of BL21 (DE3) pLysS bacteria expressing RNaseA (basic) were pelleted. Cells were lysed and inclusion bodies separated and dissolved using the ProteoSpinTM Inclusion Body Protein Isolation Micro Kit. The resulting protein was bound to the ProteoSpinTM column, washed and eluted in 50 µL of the provided elution buffer. The eluted protein samples were analyzed in 12.5% polyacrylamide gels, which were run for 45 minutes at 200 V/6.5 cm. The protein bands were made visible by staining with Coomassie Blue R-250. Figure 3: Efficient Isolation of Inclusion Body Proteins. Following gene expression, inclusion bodies were extracted, solubilized and proteins were purified using Norgen's Inclusion Body Protein Isolation Kits. Briefly, cells were pelleted and lysed using the provided Cell Lysis Reagent. The inclusion bodies were separated via centrifugation and washing and dissolved using the Inclusion Body Solubilization Reagent. Recovered proteins were analyzed on 12% SDS-PAGE and stained with Coomassie Brilliant Blue R-250. Numbers represent kDa sizes of protein bands. As can be seen, proteins of a wide range of masses are efficiently purified. Lane numbers represent size of protein purified in kDa. Figure 4: No Loss of Proteins when Isolating a Basic Protein. 100 mL of induced bacterial culture expressing a recombinant 30 kD BASIC protein were pelleted and processed using the ProteoSpinTM Inclusion Body Protein Isolation Maxi Kit. Briefly, pelleted cells were lysed, inclusion bodies were separated and subsequently dissolved using the provided Inclusion Body. Solubilization Reagent. Fractions of input, flowthrough, wash and elution were loaded on a 12.5% acrylamide gel. Lane 1 is the input, Lane 2 is the flowthrough from the input, Lane 3 is the wash, Lane 4 is the first elution and Lane 5 is the second elution. As can be seen, proteins are not lost in the flowthrough or wash. Recombinant proteins were efficiently bound to the column and eluted. Figure 5: No Loss of Proteins when Isolating An Acidic Protein. 100 mL of induced bacterial culture expressing a recombinant 70 kD ACIDIC protein were pelleted and processed using the ProteoSpinTM Inclusion Body Protein Isolation Maxi Kit. Briefly, pelleted cells were lysed, inclusion bodies were separate and subsequently dissolved using the provided Inclusion Body Solubilization Reagent. Fractions of input, flowthrough, wash and elution were loaded on a 12.5% acrylamide gel. Lane 1 is the input, Lane 2 is the flowthrough from the input, Lane 3 is the wash, Lane 4 is the first elution and Lane 5 is the second elution. As can be seen, proteins are not lost in the flowthrough or wash. Recombinant proteins were efficiently bound to the column and eluted. Next Click for expanded view Kit Specifications Maximum Culture Volume 1.5 mL Yield from 1.5 mL Up to 50 μg Minimum Elution Volume 30 μL Time to Process 12 Samples 60 minutes Storage Conditions The Cell Lysis Reagent and IB Solubilization Reagent should be stored at 4°C upon receipt of this kit. All other solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment. Once opened, the solutions should be stored at 4°C when not in use except for the pH Binding Buffers (Acidic and Basic). Some precipitation will occur with 4°C storage. This precipitation should be dissolved with slight heating to room temperature before using. Component Cat. 10300 (Micro - 25 preps) Cat. 17700 (Maxi - 4 preps) Wash Solution C 30 mL 130 mL Wash Solution N 30 mL 130 mL Binding BUffer A 4 mL 20 mL Binding Buffer N 4 mL 20 mL Elution Buffer C 8 mL 2 x 30 mL Protein Neutralizer 4 mL 4 mL Cell Lysis Reagent 15 mL 110 mL IB Solubilization Reagent 2 mL 50 mL Syringes, 1cc, slip tip 25 - Needles (Bev, 20G x 1 inch) 25 - Syringes, 10 mL, Luer-Lok™ Tip - 4 Needles (18G x 1.5 inch) - 4 Micro Spin Columns 25 - Maxi Spin Columns (filled with SiC) inserted into 50 mL collection tubes - 4 Collection Tubes 25 - Elution Tubes (1.7 mL) 25 - Elution Tubes (50 mL) - 4 Product Insert 1 1 Documentation PROTOCOLS (10300) Inclusion Body Isolation Micro Kit - Protocol (25 prep) (17700) Inclusion Body Isolation Maxi Kit - Protocol (4 prep) APPLICATION NOTES Screening For Bacterial Clones Expressing Inclusion Body Proteins A Time Course Induction Study of Recombinant Protein Expression In E Coli SAFETY DATA SHEETS (10300) Inclusion Body Isolation Micro Kit - Safety Data Sheet (17700) Inclusion Body Isolation Maxi Kit - Safety Data Sheet FLYERS Flyer RELATED BLOGS 3 Reasons why Norgen’s Industry-Leading Patented RNA Purification Technology is Best-in-Class Citations Maxi (17700) Title CP40 from Corynebacterium pseudotuberculosis is an endo-β-N-acetylglucosaminidase Journal BMC Microbiology. 2016. Authors Shadnezhad A, Naegeli A, Collin M. Title Proteome of Human Calcium Kidney Stones. Journal Urology. 2010. Authors Canales BK, Anderson L, Higgins L, Ensrud-Bowlin K, Roberts KP, Wu B, Kim IW, Monga M.
ProteoSpin™ Inclusion Body Protein Isolation Kits For the rapid isolation of inclusion body proteins For research use only and NOT intended for in vitro diagnostics. Powered by Bioz ProteoSpin™ Inclusion Body Protein Isolation Kits For the rapid isolation of inclusion body proteins Price USD$335.00 SKU 10300 Format Micro Maxi Size 25 Preps Quantity
