ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Kits
For research use only and NOT intended for in vitro diagnostics.
ProteoSpin™ Total Protein Concentration, Detergent Clean-Up and Endotoxin Removal Kits
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Supporting Data
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%.
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Kit Specifications
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| Maximum Protein Input |
200 ÎĽg
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| Maximum Column Volume Input |
600 ÎĽL
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| Molecular Weight of Recovered Proteins | No Molecular Weight cut-off |
| Final Endotoxin Levels |
≤ 0.01 EU/μg protein
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| Protein Recovery |
90-95%
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| % Detergent Removal | 90-95% |
| Detergents that can be Removed | Including Triton® X-100, CHAPS, NP-40 and Tween 20 |
| Minimum Elution Volume |
50 ÎĽL
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| Time to Complete 10 Purifications |
20 minutes
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Storage Conditions
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years from 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
FAQs
Mini
The protein solution may not be flowing through the column due to one or more of the following:
- During the binding step for Protein Concentration and Detergent Clean-Up:
- Centrifugation speed was too low.
Check the centrifuge to ensure that it is capable of generating 5,200 x g (~8,000 RPM). Sufficient centrifugal force is required to move the liquid phase through the resin. Centrifugation speeds may be increased to 14,000 x g (~14,000 RPM), but this speed should not be exceeded.
- Inadequate spin time.
Spin an additional two minutes to ensure that the liquid is able to flow completely through the column.
- Cellular debris is present in the protein solution.
Prior to the sample preparation step, filter the sample with a 0.45 µM filter or spin down insoluble materials. Solid, insoluble materials can cause severe clogging problems.
- Protein solution is too viscous.
Dilute the protein solution and adjust the pH to either 3.5 - 4 or 7 with the appropriate Binding Buffer. Highly viscous materials due to high protein concentrations can slow down flow rate significantly.
- Protein solution is not completely dissolved.
Dissolve the sample in a larger amount of buffer. Solid, insoluble materials can cause clogging problems.
- Centrifugation speed was too low.
- For Endotoxin Removal:
- Centrifugation speed was too low.
Check the centrifuge to ensure that it is capable of generating 5,200 x g (~8,000 RPM). Sufficient centrifugal force is required to move the liquid phase through the resin. Centrifugation speeds may be increased to 14,000 x g (~14,000 RPM), but this speed should not be exceeded.
- Inadequate spin time.
Spin for an additional minute to ensure that the liquid is able to flow through the column.
- Cellular debris is present in the protein solution.
Prior to the sample preparation step, filter the sample with a 0.45 µM filter or spin down insoluble materials. Solid, insoluble materials can cause clogging problems.
- Centrifugation speed was too low.
Poor peptide/protein recovery could be due to one or more of the following:
- Incorrect pH adjustment of protein sample.
It is important that the proper amount of the appropriate Binding Buffer is added to the protein sample in order to adjust the pH to either 3.5 - 4 or 7 prior to loading onto the column.
- Incorrect procedure was used.
Ensure that the acidic protocol was used for total proteins and the basic protocol was used for basic proteins.
Poor peptide recovery for Endotoxin Removal could be due to one or more of the following:
- Input protein solution is very high in molarity.
The Binding Buffer J may not lower the pH of the protein solution sufficiently if the input protein solution has a very high molarity. Dilute the molarity of the input to < 50 mM with water. If the volume of the input amount is increased over the maximum 450 µL input, the sample can be split and processed using 2 columns.
- The appropriate amount of Binding Buffer J was not added.
Ensure that 20 µL of Binding Buffer J is added for every 500 µL of protein processed. The protein volume must not exceed 450 µL.
The eluted protein may be degraded due to one or more of the following:
A) For Protein Concentration and Detergent Clean-Up:
- Eluted protein solution was not neutralized.
Add 9.3 µL of Neutralizer to each 100 µL of eluted total proteins in order to adjust the pH to neutral. Some proteins are sensitive to high pH, such as the elution buffer at pH 12.5.
- Eluted protein was not neutralized quickly enough.
If eluted proteins are not used immediately, degradation will occur. We strongly suggest adding Neutralizer in order to lower the pH.
- Proteases may be present.
Use protease inhibitors during all steps of the Sample Preparation.
- Bacterial contamination of protein solution.
Prepare the protein sample with 0.015% sodium azide.
B) For Endotoxin Removal:
- Eluted protein was not neutralized.
Add 9.3 µL of Protein Neutralizer EF EF for every 100 µL of eluted protein in order to adjust the pH to neutral. Some proteins are sensitive to high pH, such as the Elution Buffer G.
- Bacterial contamination of protein solution.
The protein samples can be prepared with 0.015% sodium azide.
- Eluted protein was not neutralized quickly enough.
If the eluted protein is not neutralized immediately, degradation will occur. We strongly recommend adding Protein Neutralizer EF EF in order to lower the pH.
If the protein does not perform well in downstream applications for endotoxin removal, it may be because a different Elution Buffer was used. The provided Elution Buffer G has been optimized for endotoxin-free recoveries. The endotoxin-free properties of the eluted protein will be compromised if another Elution Buffer G is used. If a different Elution Buffer G other than the one provided is used, the buffer should also be checked for any components that may interfere with the application. Common components that are known to interfere are high salts, detergents, and other denaturants. Check the compatibility of your Elution Buffer with the intended use.
If endotoxin levels are high in the elution after the endotoxin removal protocol, it may be caused by:
- Using a different Elution Buffer.
The provided Elution Buffer G has been optimized for endotoxin-free recoveries. The endotoxin-free properties of the eluted protein will be compromised if another Elution Buffer is used. If a different Elution Buffer other than the one provided is used, the buffer should also be checked for endotoxin levels.
- Extremely high endotoxin levels in input protein.
Check the endotoxin levels of your protein input. The endotoxin levels of the eluted protein may be reduced significantly compared to the input.