| Product Description | Aladdin's UltraBio™ Anti-Flag Magnetic Beads are based on amino magnetic beads, with 200nm particle size, covalently coupled with high-quality mouse monoclonal antibody that recognizes the Flag octapeptide sequence (DYKDDDDK). This product can specifically bind with Flag-tagged proteins expressed in animals, plants, and microorganisms, and is recommended to use for immunoprecipitation (IP), co-immunoprecipitation (Co-IP), and protein purification.The Flag-tag is an octapeptide consisting of 8 amino acid residues (DYKDDDDK). The commonly used forms are Flag and 3X Flag, which can be fused either to the N-terminus or the C-terminus of a target protein to facilitate protein purification and immunoassays. Flag-tag has the following advantages
Precautions： Repeated freezing and thawing of this product up to 3 times does not cause obvious changes in its performance.The pH of Mag™ Anti-Flag Magnetic Beads should be maintained at 6-8. Do not centrifuge, dry, or freeze the magnetic beads. Long-time exposure of the beads to a magnetic field will causes beads to aggregate.Resuspend Mag™ Anti-Flag Magnetic Beads evenly in solution by gentle pipetting prior to use. Do not vortex.We recommend including both negative and positive controls when performing IP assays.Protein samples should be purified after collection as soon as possible and kept at 4ºC or on ice to minimize protein degradation or denaturation. Protein degradation can also be inhibited by adding appropriate protease inhibitors (, P1005/P1006, P1048/P1049, P1010/P1011, P1050/P1051).If using a vacuum pump to aspirate supernatant, the strength of the vacuum pump should be controlled properly to avoid the aspiration of magnetic beads.It is normal to see magnetic beads aggregate when an acidic solution is used for the elution, which can be prevented effectively by containing 0.1% non-ionic detergent (such as Triton X-100, Tween-20, or NP-40) without affecting the performance of this product.High concentrations of DTT, mercaptoethanol, or anidin hydrochloride might affect the binding of the target protein to this product, but it is compatible with ’s Cell Lysis Buffer for Western and IP , RIPA Lysis Buffer or NP-40 Lysis Buffer . For detailed information about the Lysis Buffers produced by , please refer to the following webpage:http://www.aladdin-e.com/support/lysis-buffer.htm.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use： 1. Preparation of protein samplesa. Lysis cells or tissues with appropriate lysis buffer. We recommend using 's Cell Lysis Buffer for Western and IP as a priority. Under certain circumstances, try ’s RIPA Strong Lysis Buffer , RIPA Medium Lysis Buffer , or RIPA Weak Lysis Buffer . Other lysis buffers with a pH of 6-8 can also be used.b. After lysis and centrifuge, keep the supernatant at 4℃ or on ice for subsequent use. We recommend performing the subsequent procedures on the same day as the protein sample is prepared. Otherwise, make aliquots and keep them at -80℃ for future use.2. Preparation of Anti-Flag Magnetic Beadsa. Resuspend the Magnetic Beads in the vial (gently pipette for 10 times, do not vortex). Transfer 10-20μl of Magnetic Beads suspension into a new tube (for 500μl of protein sample). The amount of beads suspension can be scaled up or down proportionally based on the volume of the protein sample.Note: If using more than 0.2 ml of bead suspension, place the tube into a magnetic stand to collect beads and remove the supernatant.b. Add 500μl of 1X TBS (, ST661/ST665) to the beads and pipette gently to mix. Place the tube into a magnetic stand to collect the beads against the side of the tube. Remove and discard the supernatant. Repeat this step twice.c. Resuspend the Magnetic Beads with 1X TBS at an equal volume to the initial volume of beads suspension taken in step a (e.g., if 10μl of beads suspension is taken, add 10μl of 1X TBS).3. Protein bindinga. Add 500μl of cell lysate to the washed beads, pipette gently to resuspend beads, and incubate for 2 hours at room temperature or overnight at 4ºC while gently rotating the tube on a rotary mixer.Note: Occasional aggregation of magnetic beads during the binding process doesn’t affect experimental results.b. Place the tube into a magnetic stand to collect beads against the side of the tube. Remove and discard the supernatant. Note: A small amount of supernatant can be reserved in a clean EP tube for examination of the binding results.c. Wash beads with 500μl of 1X TBS and gently pipette to mix. Place the tube into a magnetic stand to collect the beads against the side of the tube. Remove and discard the supernatant. Repeat this step 3 times.Note: The A280 of supernatant can also be measured to determine whether the beads are washed thoroughly. Repeat washing until the A280 is smaller than 0.05. 4. ElutionBased on the features of the target protein or further usage, one of the following three elution methods can be used.a. Competitive elution with 3X Flag under native conditions. This elution method has a high elution efficiency, and the eluted protein maintains its original bioactivity, which is beneficial for subsequent analysis. (a) Preparation of 3X Flag elution buffer. Dissolve 3X Flag Peptide (, P9801-25mg) in 1X TBS buffer to a final concentration of 150μg/ml, or dilute the 3X Flag Stock Solution at 5mg/ml (, P9801) with 1X TBS buffer to a final concentration of 150μg/ml.(b) Add 100μl of 3X Flag elution buffer to resuspend the beads by pipetting gently and incubate with gentle shaking or on a rotator for 30-60 minutes at room temperature or 1-2 hours at 4℃. The elution efficiency can be improved by prolonging the incubation time appropriately or by repeated elution.(c) Place the tube into a magnetic stand for 10 seconds and transfer the supernatant to a clean EP tube. (d) For immediate use, keep the eluates at 4℃, or store at -20℃ or -80℃ for long-term storage. b. Elution under acidic conditions. This method is rapid and highly efficient. The eluted protein also maintains its original bioactivity, which is beneficial for subsequent analysis.(a) Preparation of acidic elution buffer (0.1M Glycine-HCl, pH3.0) and neutralization buffer (0.5M Tris-HCl, pH7.4, 1.5M NaCl).(b) Add 100μl of acidic elution buffer to resuspend the beads by gently pipetting and incubate with gentle shaking or on a rotator for 5 minutes at room temperature (no more than 15 minutes).(c) Place the tube into a magnetic stand for 10 seconds and transfer the supernatant to a new EP tube. Add 10μl of neutralization buffer immediately to neutralize the low pH, which helps preserve the bioactivity of the target protein. Mix well gently. (d) For immediate use, keep the eluates at 4℃, or store at -20℃ or -80ºC for long-term storage. (e) To obtain higher elution efficiency, repeat steps b-c and combine the eluates.Note 1: The elution efficiency of the acidic elution method might be lower than the other two elution methods.Note 2: The elution efficiency of the acidic elution method depends on the features of the target protein. To obtain a higher elution efficiency, the pH of the acidic elution buffer can be optimized from 2.5 to 3.1. The pH or amount of neutralization buffer needed to neutralize the eluates should also be adjusted appropriately. c. Elution with 1X SDS-PAGE loading buffer under denaturing conditions. The eluate can be used for gel electrophoresis and immunoblotting. (a) Preparation of 1X SDS-PAGE loading buffer. The SDS-PAGE loading buffer can be homemade or purchased from . The conventional SDS-PAGE loading buffer contains reducing reagents, such as DTT, that divides antibody into heavy and light chains. (b) Add 100μl of 1X SDS-PAGE loading buffer to resuspend the magnetic beads and boil for 5 minutes. (c) Cool and place the tube into a magnetic stand for 10 seconds and transfer the supernatant to a new tube. Keep the eluates for SDS-PAGE or Western analysis.FAQ: ProblemPossible CausesSolutionsVery few or no Flag‐tagged protein in the eluate.Protein is not completely eluted.Use a different elution method.No target protein expressed.Make sure the target protein has Flag-tag by Western blot or dot blot analyses.Very low protein expression level.1. Use more cells or tissues.2. Optimize expression conditions to increase the expression level of the target protein.Washes are too stringent.Reduce the duration and number of washes.Incubation time is inadequate.Prolong the incubation time.Interfering substances present in the sample.Lysate contains high concentrations of DTT, 2-mercaptoethanol, or other reducing agents that may interfere with antigen-antibody interaction.Low sensitivity of the detection system.If Western blot detection is used:1. Check the specificity of primary and secondary antibodies using proper controls.2. Check the protein transfer efficiency by using a prestained protein marker or staining the membrane with Ponceau S.3. Use fresh ECL western substrate or try a different ECL substrate with higher sensitivity.The background is too high.Nonspecific binding of proteins to antibodies, beads, or EP tubes.1. Pre-clear lysate with Mouse IgG Magnetic Beads to remove nonspecific binding proteins.2. After suspending beads for the final wash, transfer the entire sample to a clean microcentrifuge tube before centrifugation.Insufficient washing of magnetic beads.1. Increase the number of washes.2. Prolong the duration of each wash to at least 15 minutes.3. Increase the salt and/or detergent concentrations in the wash buffer4. Centrifuge at a lower speed to avoid nonspecific trapping of denatured proteins.Multiple protein bands found in the eluate.The protein is not stable at room temperature.Purify the target protein at lower temperatures, such as 4ºC.Protein degradation due to protease activity during the purification process.Add appropriate protease inhibitors to the cell lysate.Non‐specific binding.1. Prepare cell lysate again.2. Add additional wash steps.
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