Visit our interactive pathways at /pathways User Protocol 488250 Rev. 5 January 2006 RFH Page 1 of 1 Non-Interfering Protein Assay Kit Cat. No. 488250 Note that this user protocol is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions. Full details are available at. Introduction The Non-Interfering Protein Assay TM has been extensively tested to work in the presence of common laboratory agents, such as: reducing agents (2ME, DTT), chelating agent -EDTA, detergents (non-ionic, anionic, cationic, and zwitterionic), amines (Tris), sugar, urea, ammonium sulfate, guanidine hydrochloride, guanidine thiocyanate, drugs, antibiotics, cobalt, and numerous other agents and extraction buffers. Principle behind the Non-Interfering Protein Assay TM : 1. Non-Interfering Protein Assay TM uses a Universal Protein Precipitating Agent "UPPA" (Patent Pending) for removing interfering agents from protein solutions. The UPPA reagent when mixed with protein solutions rapidly precipitates and immobilizes protein in the sample tube. This allows removal of interfering agents present in the protein solution. Clean protein is all that remains in the assay tube. 2. Non-Interfering Protein Assay TM is substantially independent of the protein side chain groups. This assay is based on the specific binding of copper ions to the peptide backbone of protein. Protein solution is mixed with an alkaline solution containing a predetermined concentration of copper salt; the copper ions bind with the peptide backbone of the protein leaving unbound copper ions behind in the solution. The unbound copper is measured with a color-producing agent. As the concentration of protein increases, the concentration of unbound copper decreases, thus the color density is inversely related to the amount of protein present in solution. Therefore, the Non-Interfering Protein Assay TM in effect measures unbound copper ions present in protein solution and is not be affected by protein side chains groups. This approach allows the Non-Interfering Protein Assay TM to overcome protein-to-protein variations. The combined strategy of removing interfering agents from the protein solution and the fact that Non- Interfering Protein Assay TM measures unbound copper in protein solution allows a highly reliable determination of protein concentration. Assay sensitivity: The assay has been standardized to provide a linear response between 0-50 µg of protein in the assay volume. This assay is ideal for micro assay protocols. The assay has a sensitivity threshold of 0.5 µg protein in assay volume. Kit Components: Suitable for 500 Assays Containing Universal Protein Precipitating Agent (UPPA) UPPA-I UPPA-II Copper Solution-Reagent-I Color Agent-A Color Agent-B Protein Standard BSA (2 mg/ml) Suitable for 500 Assays 250 ml 250 ml 50 ml 2 x 250 ml 5 ml 5 ml merckbiosciences.de
User Protocol 488250 Rev. 5 January 2006 RFH Page 2 of 2 Storage conditions: Store UPPA-I and UPPA-II at room temperature. The rest of the kit should be stored in the dark and refrigerated in its original box. Always use clean, sterile pipets and aseptic techniques for removing reagents from the reagent bottles. When stored properly, the kit is stable for one year. TOLERANCE TO COMMON LABORATORY AGENTS: The protein assay shows wide tolerance to both ionic and non-ionic detergents such as SDS, Triton-X100, CHAPS, phosphate buffers, urea, guanidine HCl, and other common laboratory agents. The following table includes a few compounds that have been tested for interference in this assay. Compounds Tested Concentration Interference Triton -X 100, -X114 detergent 3% No CHAPS 1% No CHAPSO 1% No Tween -20 detergent 2% No Tesit 2% No Brij 35 detergent 1% No SDS 1% No Sarcosyl 1% No N-Octyl Glucoside 0.5% No Digitonin 0.3% No Ammonium Sulfate 40% No Urea 8M No Guanidine HCl 6M No Guanidine Thiocyanate 4M No Tris/HCl 0.2M No HEPES 0.1M No Phosphate buffer 0.2M No EDTA 10mM No 2-Mercaptoethanol 0.5% No DTT 0.1mM No Glycerol 30% No Sucrose 30% No Sodium azide 0.1M No EXTRACTION BUFFERS Containing [4M Urea, 1% SDS, 10mM EDTA, and 0.8% 2-Mercaptoethanol] No Containing [1% Sarcosyl, 2 Mercaptoethanol, 4M Guanidine Thiocyanate, and 10 mm EDTA] No PROTOCOLS PREPARATION BEFORE USE: Prepare Reagent II by mixing color agents A and B: Prior to use, prepare an appropriate volume of Reagent-II by mixing 100 parts of Color Agent-A with 1 part of Color Agent-B. Mark this reagent solution as Reagent II. Example: For making 10 ml of Reagent II: Mix 10 ml of Color Agent-A and 0.1 ml of Color Agent-B in a clean tube. Fresh or unused Reagent II can be stored refrigerated and is good for one month or as long as the optical density of the solution remains below 0.025 ABS at 480 nm. PROTOCOL 1: Suitable for 2ml Assay Tubes Read "PREPARATION BEFORE USE" 1. Perform assays at room temperature. Use 2 ml tubes for assay. 2. Prepare a calibration curve using the BSA standard supplied with the kit. The reaction volume for the assay is 1.5 ml that is suitable for most spectrophotometer cuvettes. However, if needed the total reaction volume can be adjusted by changing the volume of the reagent solutions proportionately. merckbiosciences.de
User Protocol 488250 Rev. 5 January 2006 RFH Page 3 of 3 3. Prepare a blank without any BSA standard. In a series of tubes, add 1-25 µl of 2 mg/ml BSA standard (see Example #1). If you are using Pre-Diluted Protein Standard, add 50 µl from each vial (0.1-1.0 mg/ml) 4. Add 1-50 µl of the samples to be assayed in duplicate tubes. NOTE: The total amount of protein added in any assay tube should not exceed 40-50 µg/tube. If necessary, dilute the test samples in water before performing assay. 5. Add 500 µl UPPA-I into each tube, and vortex the tube. Incubate the tubes for 2-3 minutes at room temperature. 6. Add 500 µl UPPA-II into each tube and mix the contents of each tube. Centrifuge the tube at 5-10,000 x g or at maximum speed to sediment the precipitate. 7. Discard the supernatant by inverting the tube on a clean absorbent tissue paper. Allow the liquid to completely drain from the tube. 8. OPTIONAL: Read optional precipitate washing step in TROUBLE SHOOTING section below. 9. Add 100 µl Copper Solution Reagent-I and 400 µl de-ionized water into each tube and mix to dissolve the protein precipitate. 10. Add 1 ml Reagent-II into each tube and mix instantaneously. For instantaneous mixing, Reagent-II should be introduced by rapidly shooting in to the tube. Absorbance can be read after 15-20 minutes. 11. Read absorbance of your sample at 480 nm against water as the reference. The absorbance should be read within 30-60 minutes. 12. Plot absorbance against protein concentration to generate your standard curve, and determine protein concentrations of unknowns in relation to your standard curve. 13. NOTE: Do not subtract blank reading from the sample reading. Absorbance will decrease as protein concentration increases. PROTOCOL-1 SUMMARY: Example-1 Tube # 1 2 3 4 5 6 7 8 2 mg/ml BSA Standard (µl) 0 4 8 12 20 25 - - Protein (µg) 0 8 16 24 40 50 - - Test Sample (µl) - - - - - - 50(?) 50(?) Add 500 µl UPPA-I into each tube. Mix and incubate for 2-3 minutes. Add 500 µl UPPA-II into each tube, mix and centrifuge to sediment protein precipitate. Discard supernatant by inverting the tubes on a clean absorbent tissue paper. Allow the liquid to completely drain off the tube. Add Copper Solution Reagent-I: 100 µl into each tube and then add 400 µl water into each tube and mix. Add Reagent-II: 1 ml into each tube and mix instantaneously. Read absorbance at 480 nm PROTOCOL 1: Suitable for High Throughput Assay using 96 - Well Plates Read "PREPARATION BEFORE USE" 1. For best results we recommend use of 2 ml deep well titer plates - round or V-bottom. These plates are available through various sources, such as VWR or Fisher or by comparable distributors elsewhere. 2. Plate Protocol requires centrifugation of plates. Make sure you have access to a centrifuge capable of centrifuging titer plates. 3. Use the standard Protocol-1 (above) for 2 ml Protein Assay Tubes. Except in Step-6, centrifuge the plate at 2-5,000 x g or at a maximum speed, for 5-7 min, to sediment the precipitate. 4. Discard the supernatant by inverting the titer plate. Allow the liquid to completely drain off the wells by resting the inverted titer plate on a clean absorbent tissue paper. 5. Follow rest of the Protocol as above. 6. For reading the absorbance of each reaction well, transfer 0.2-0.25 ml of assay reaction product from each well into the corresponding well of a new flat bottom plate. Read absorbance at 480 nm within 30-60 min against water. Plot absorbance against protein concentration, and determine protein concentration of unknowns. merckbiosciences.de
User Protocol 488250 Rev. 5 January 2006 RFH Page 4 of 4 NOTE: Do not subtract blank reading from the sample reading. Absorbance will decrease as protein concentration increases. PROTOCOL 2: Suitable when interference to protein assay is not a concern. Suitable when protein is in water or other non-interfering solutions. This protocol does not use the protein precipitation step to remove interfering agents. Read PREPARATION BEFORE USE 1. Perform assays at room temperature. 2. Prepare a calibration curve using the BSA standard supplied with the kit. The reaction volume for the assay is 1.5 ml, which is suitable for most spectrophotometer cuvettes. However, if needed the total reaction volume can be adjusted by changing the volume of the reagent solutions proportionately. 3. In a series of tubes add 1-25 µl of 2 mg/ml BSA standard (see Example-2). Prepare a blank without any BSA standard. If you are using Pre-Diluted Protein Standard, add 50 µl from each vial (0.1-1.0 mg/ml) 4. Add 1-50 µl of test samples in duplicate tubes. NOTE: The total amount of protein added in any tube should not exceed 40-50 µg/tube. If necessary, dilute the test samples in water before performing assay. 5. Add deionized water into each tube to bring the total volume to 400 µl. 6. Add 100 µl Copper Solution Reagent-I to each tube and mix. 7. Add 1000 µl Reagent-II into each tube and mix instantaneously. For instantaneous mixing, Reagent-II should be introduced by rapidly shooting in to the tube. Absorbance can be read after 15-20 min. 8. Read absorbance at 480 nm against water. The absorbance should be read within 30-40 min. 9. Plot absorbance against protein concentration to generate your standard curve, and determine protein concentrations of unknowns in relation to your standard curve. NOTE: Do not subtract blank reading from the sample reading. Absorbance will decrease as protein concentration increases. PROTOCOL-2 SUMMARY: Example-2 Tube # 1 2 3 4 5 6 7 8 2 mg/ml BSA Standard, (µl) 0 4 8 12 20 25 - - Protein (µg) 0 8 16 24 40 50 - - Test Sample (µl) - - - - - - 50(?) 50(?) Add water to make 400 µl 400 396 392 388 380 375 350 340 Reagent-I (µl) 100 100 100 100 100 100 100 100 Reagent-II (µl) 1000 1000 1000 1000 1000 1000 1000 1000 PROTOCOL 2: Suitable for High Throughput Assay using 96 - Well Plates Read PREPARATION BEFORE USE instruction of standard protocol. 1. For the best result we recommend use of 2 ml deep well plates-round or V-bottom. These plates are available through various sources, such as VWR or Fisher or by comparable distributors elsewhere. 2. Follow the Protocol-2 (above) for 96 well plate as above. 3. For reading the optical density of each reaction well, transfer 0.2-0.25 ml assay reaction product from each well into a reading titer plates (0.2-0.25 ml) with flat bottom. Absorbance may also be read manually or using robotic sampler and auto-readers. 4. Read absorbance at 480 nm against water. The absorbance should be read within 30-60 min. Plot absorbance against protein concentration, and determine protein concentrations of unknowns. NOTE: Do not subtract blank reading from the sample reading. Absorbance will decrease as protein concentration increases. merckbiosciences.de
User Protocol 488250 Rev. 5 January 2006 RFH Page 5 of 5 TROUBLE SHOOTING 1. If the results with the Non-Interfering assay are unsatisfactory or showing signs of interference, the assay may be contaminated with interfering agents present in protein solution. To over come the carry-over problems, introduce a precipitate washing step as follows: After UPPA-I and II treatments, centrifuge and discard the supernatant. Repeat the UPPA I and II treatment by adding 0.5 ml UPPA-I into each tube and quickly adding 0.1 ml UPPA-II. Gently invert the tube a few times without disturbing the precipitate (for 96 well plates agitate the plate) and centrifuge for 5 minutes. Discard the supernatant and follow the remainder of the protocol. 2. The absorbance of the tube without protein should be approximately two times higher than the tube containing 50 µg protein. If the slope of the plot is shallow, make sure that the Reagent-II is introduced into the tube or well and mixed instantaneously. Trademarks Calbiochem is a registered trademark of EMD Biosciences, Inc. Triton is a registered trademark of Dow Chemical Company Brij and Tween are registered trademarks of ICI Americas, Inc. Prices and availability are subject to change. Copyright 2005 EMD Biosciences, Inc., an affiliate of Merck KGaA, Darmstadt,. All rights reserved. Each product is sold with a limited warranty which is provided with each purchase. Each product is intended to be used for research purposes only. It is not to be used for drug or diagnostic purposes nor is it intended for human use. EMD Biosciences products may not be resold, modified for resale, or used to manufacture commercial products without written approval of EMD Biosciences. merckbiosciences.de