ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Kits For rapid and efficient endotoxin removal from proteins and peptides For research use only and NOT intended for in vitro diagnostics. Powered by Bioz ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Kits For rapid and efficient endotoxin removal from proteins and peptides SMART Save USD$179.00 USD$143.20 SKU 22200 Format Mini Maxi Size 4 Preps Quantity Overview Details Documentation Citations Overview Columns bind proteins of interest while endotoxins flow through Reduce endotoxin levels to less than 0.01 EU/µg of protein Proteins are desalted Greater than 95% protein recovery Concentrate protein samples and remove detergents at the same time Effectively remove a wide range of detergents including SDS, Triton® X-100, CHAPS, NP-40, and Tween 20 Purification is based on spin column chromatography that uses Norgen’s resin separation matrix The ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Kits are designed for the rapid removal of endotoxins from previously purified proteins or peptides, with protein recoveries of > 95% being achieved. Endotoxins, also known as lipopolysaccharides, are cell-membrane components of Gram-negative bacteria such as E. coli. Endotoxins liberated by Gram-negative bacteria are frequent contaminations of protein solutions derived from bioprocesses. Due to the high toxicity of endotoxins in vivo and in vitro, their removal from protein preparations is often necessary prior to the use of the protein in downstream applications. These kits efficiently reduce endotoxin levels to ≤ 0.01 EU/mg of protein, using spin column chromatography based on Norgen’s proprietary resin as the separation matrix. These kits are highly efficient in removing many different salts commonly used in the laboratory including, but not limited to, MgCl2, NaCl, KCl, CaCl2, LiCl and CsCl. The purified protein samples can be used in a number of downstream applications including sequencing, cloning, and in vitro and in vivo introduction into cells and organisms for various purposes. The simultaneous removal of salts while concentrating a dilute protein solution makes the kit a convenient method for preparing proteins before running many downstream applications such as SDS-PAGE, isoelectric focusing, X-ray crystallography, NMR spectroscopy, mass spectroscopy and other applications. Mini Kit The ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Mini Kit is designed for the rapid removal of endotoxins from up to 200 μg of previously purified proteins or peptides, with protein recoveries of > 95% being achieved. Maxi Kit The ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Maxi Kit is designed for the rapid removal of endotoxins from up to 4 mg of previously purified proteins or peptides, with protein recoveries of > 95% being achieved. Details Supporting Data Previous Figure 1. Endotoxin-Free Proteins. Endotoxins were removed from 100 µg protein samples in triplicate using theProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Mini Kit. Samples containing 100 µg of BSA were spiked with endotoxins (0.9 EU/mg). The endotoxin-spiked BSA was then cleaned, in triplicate, using the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Mini Kit by using 5 µL of the provided Endotoxin Removal Solution in the presence of isopropanol. On average, the endotoxin levels in the 100 µg samples were reduced from 0.9 EU/mg to 0.005 EU/mg. Figure 2. High Protein Recovery. An average protein recovery of greater than 95% was achieved when endotoxins were removed from 100 µg inputs of protein in triplicate. Samples containing 100 µg of BSA were spiked with endotoxins (0.9 EU/mg). The endotoxin-spiked BSA was then cleaned, in triplicate, using the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Mini Kit by using 5 µL of the provided Endotoxin Removal Solution in the presence of ethanol. The BioRad Protein Assay was used to determine the amount of proteins present in the elutions, and 91 µg, 99 µg and 89 µg of BSA were recovered after the removal of the spiked endotoxins. There is very little loss of protein associated with using the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Mini Kit. Figure 3. Efficient Removal of SDS from Lysate. SDS was removed from various types of lysates (DH5α for bacterial lysate and HeLa cells for mammalian lysates) prepared with either 1.0% or 0.1% SDS using the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Mini Kit. The Genotech SDS-Out Kit was used to measure the concentration of the SDS after cleaning. SDS removal was found to be greater than 99% in all cases. Figure 4. Examples of BSA Volume Concentration using the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Mini Kit.To demonstrate the ability of the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Mini Kit to concentrate protein samples, 50 µg of BSA was spiked into 1, 3 and 5 mL of a 50 mM sodium acetate solution (pH 4.5). The samples were processed as per the provided protocol, and all samples were eluted into 50 µL of the provided Elution Buffer. The 1 mL input was therefore concentrated 20X, the 3 mL input was concentrated 60X, and the 5 mL input was concentrated 100X. Figure 5. Examples of BSA Concentration using the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Mini Kit. To further demonstrate the ability of the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Mini Kit to concentrate protein samples, 50 µg of BSA was spiked into 2 mL of a 50 mM sodium acetate solution (pH 4.5). The sample was processed as per the provided protocol, and was eluted into 50 µL of the provided Elution Buffer. The amount of recovered protein was then determined using the BioRad Protein Assay. It was found that 48 µg of the input protein was recovered, corresponding to a 38X protein concentration based on mass. Figure 6. Representative Recovery and Salt Reduction using the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Mini Kit. To investigate salt removal using the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Mini Kit, 50 µg samples of BSA were prepared in 1 mL samples of sodium acetate and were spiked with 300 ppm of MgCl2, NaCl, KCl, CaCl2 or LiCl. The salt was removed using the provided protocol, and the proteins were eluted into 2 separate 50 µL elutions of 250 mM ammonium hydroxide. Ammonium hydroxide is not the provided elution buffer, but was required to allow for the analysis of the amount of salt in the elutions. The 2 elutions were combined for analysis. The amount of BSA recovery was determined using the BioRad Protein Assay, and the amounts can be seen in the table above. The BSA recoveries were lower than expected due to the fact that an alternate Elution Solution was used for analysis that was not optimized for recovery. The amount of salt present in the elutions was determined by ICP/MS designed for elemental analysis. As it can be seen, salt removal was up to 99.7% using this kit. Figure 7. Efficient Removal of SDS Removal of SDS with the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Mini Kit is greater than 99%. To evaluate this, 50 µg samples of BSA were spiked with 0.1% SDS and processed using the kit. The protocol was followed, and the proteins were bound, washed and eluted as described. The purified protein was then assayed for SDS using the Detergent-OUT kit by Geno Technologies, Inc, and SDS removal was found to be greater than 99%. Figure 8. High Protein Recovery in the Presence of Detergents. To investigate the recovery of proteins in the presence of different amounts of detergent, 50 µg samples of BSA were prepared in solution and spiked with increasing amounts of SDS (0%–1.0%). The protein samples were then purified using the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Mini Kit according to the bind, wash and elution procedure to remove the SDS. The percentage of recovery of BSA was then determined using the Bradford Assay. High protein recovery was obtained even when the original SDS concentration was as high as 1%. Figure 9. Endotoxin-Free Proteins. Endotoxins were removed from 1 mg protein samples in triplicate using the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Maxi Kit. Samples containing 1 mg of BSA were spiked with endotoxins (2.3 EU/mg). The endotoxin-spiked BSA was then cleaned, in triplicate, using the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Maxi Kit by using 50 µL of the provided Endotoxin Removal Solution in the presence of Isopropanol. On average, the endotoxin levels in the samples were reduced to 0.07 EU/mg of protein. Figure 10. High Protein Recovery. An average protein recovery of greater than 95% was achieved when endotoxins were removed from 0.5 mg of protein in triplicate. There is very little loss of protein associated with using the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Maxi Kit. Figure 11. Efficient Removal of SDS. Removal of SDS with the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Maxi Kit is greater than 99%. To evaluate this, 2 mg samples of BSA were spiked with 0.1% SDS and 1% SDS, and were processed using the kit. The protocol was followed, and the proteins were bound, washed and eluted as described. The purified protein was then assayed for SDS using the Detergent-OUT kit by Geno Technologies, Inc, and SDS removal was found to be greater than 99%, with up to 50 mg of SDS being removed. Figure 12. High Recovery of Proteins from an SDS-Containing Solution. The recovery of both acidic and basic proteins from solutions containing SDS is high using the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Maxi Kit. To evaluate recovery, 10 mL buffer samples were spiked with lysozyme or BSA as well as either 0.1% SDS or 1.0% SDS as shown in the table above. The samples were processed using the kit as per the provided protocol to remove detergents, and protein recovery was assessed. Recoveries of up to 100% were observed, indicating that high protein recoveries can be achieved even in the presence of high amounts of detergents. Figure 13. Examples of BSA Volume Concentration using the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Maxi Kit. To demonstrate the ability of the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Maxi Kit to concentrate protein samples, 8 mg of BSA was spiked into 20, 60 and 100 mL of a 50 mM sodium acetate solution (pH 4.5). The samples were processed as per the provided protocol, and all samples were eluted into 4 mL of the provided Elution Buffer. The 20 mL input was therefore concentrated 5X, the 60 mL input was concentrated 15X, and the 100 mL input was concentrated 25X. Figure 14. Examples of BSA Mass Concentration using the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Maxi Kit. To further demonstrate the ability of the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Maxi Kit to concentrate protein samples, 8 mg of BSA was spiked into 20 mL of a 50 mM sodium acetate solution (pH 4.5). The sample was processed as per the provided protocol, and was eluted into 4 mL of the provided Elution Buffer. The amount of recovered protein was then determined using the BioRad Protein Assay. It was found that 7.89 mg of the input protein was recovered in 4 mL, corresponding to a 5X protein concentration based on mass. Figure 15. High Protein Recovery. Increasing amounts of BSA (1, 2, 4, 6 and 8 mg) were spiked into 20 mL of a 50 mM sodium acetate solution, and were concentrated using the ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Maxi Kit. The provided protocol was followed, and the concentrated BSA was eluted into 2 separate 4 mL elutions using the provided Elution Buffer. The elutions were then combined, and the amount of protein recovered in each elution was determined using the BioRad Protein Assay. The ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Maxi Kit allows for high protein recoveries of up to 90%. Next Click for expanded view Kit Specifications Maximum Protein Input 4 mg Minimum Protein Input 0.25 mg Maximum Column Volume Input 18 mL Molecular Weight of Recovered Proteins No Molecular Weight cut-off Final Endotoxin Levels ≤ 0.01 EU/μg protein Protein Recovery 90-95% % Detergent Removal 90-95% Detergents that can be Removed Including Triton® X-100, CHAPS, NP-40 and Tween 20 Elution Volume 2-4 mL Time to Complete 10 Purifications 30-40 minutes Storage Conditions All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment. Component Cat. 22800 (25 preps) Cat. 22200 (4 preps) Binding Buffer J 8 mL 8 mL Binding Buffer N 4 mL 20 mL Wash Solution M 50 mL 130 mL Wash Solution CIP 20 mL 60 mL Wash Solution N 30 mL 130 mL Wash Solution NIP 20 mL 60 mL Elution Buffer G 6 mL 20 mL Endotoxin Removal Solution 1.5 mL 1.5 mL Protein Neutralizer EF 2 mL 2 mL Maxi Spin Columns (assembled with Collection Tubes) - 4 Mini Spin Columns 25 - Collection Tubes 25 - Maxi Spin Columns (assembled with Collection Tubes) 4 Elution Tubes (1.7 mL) 25 - Elution Tubes (50 mL) 4 - Product Insert 1 1 Documentation PROTOCOLS (22800) ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Mini Kit - Protocol (25 preps) (22200) ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Maxi Kit - Protocol (4 prep) APPLICATION NOTES Rapid Concentration of Dilute Protein Solutions Using the ProteoSpin™ CBED Kit Rapid Desalting of Protein Solutions Using the ProteoSpin™ CBED Kit Efficient Removal of Both Free and Tightly-Bound SDS from Protein Solutions Efficient Removal of Triton X-100 Prior to MALDI-TOF and Digestion SAFETY DATA SHEETS (22800) ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Mini Kit - Safety Data Sheet (22200) ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Maxi Kit - Safety Data Sheet FLYERS Flyer Flyer RELATED BLOGS 3 Reasons why Norgen’s Industry-Leading Patented RNA Purification Technology is Best-in-Class Citations Mini (22800) Title The emetic activity of staphylococcal enterotoxins, SEK, SEL, SEM, SEN and SEO, in a small emetic animal model, house musk shrew Journal Microbiology and Immunology. 2017. 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