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 ofthe 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%.