| Product Description | Aladdin's UltraBio™ Anti-MBP Magnetic Beads are nano-scale amino magnetic beads covalently coupled with high-quality MBP mouse monoclonal antibodies. These beads can specifically bind MBP fusion proteins in lysates, serum, ascites, or other samples from animals, plants, or microorganisms. This product is mainly used for immunoprecipitation (IP), co-immunoprecipitation (Co-IP), or purification of MBP-tagged proteins or their protein complexes.Maltose binding protein (MBP) encoded by the malE gene is a component of the E. coli maltose/maltodextrin system and regulates the uptake and catabolism of maltodextrins, with a molecular weight of approximately 42kDa [1]. The MBP tag can be fused to the N- or C-terminus of a protein, with the N-terminus being the most common fusion site. The MBP tag has several advantages
Precautions： Due to the large molecular weight of the MBP tag itself (approximately 42kDa), the larger fusion protein structure may affect the recognition of the MBP tag by the Anti-MBP Magnetic Beads. We recommend performing a preliminary immunoprecipitation test to verify the effectiveness. The lysis conditions can be adjusted appropriately if necessary.Due to the strong binding affinity between the MBP tag and the MBP antibody, acidic elution may not be effective. It is recommended to use the SDS-PAGE loading buffer elution method in priority.This product should be maintained at pH of 6-8. Avoid high-speed centrifugation and drying of the magnetic beads. Long time exposure of the beads to magnetic field will cause beads to agglomerate.Properly resuspend the magnetic beads before use by gently inverting the tube several times. Do not vortex or shake the beads vigorously to avoid antibody denaturation.When performing immunoprecipitation or purification with this product, it is recommended to include both positive and negative controls.Protein samples should be purified as soon as possible after collection and should always be placed at 4℃ or on ice to minimize protein degradation or denaturation. Protein degradation can also be inhibited by adding appropriate protease inhibitors, such as Protease Inhibitor Cocktail for General Use , Protease and Phosphatase Inhibitor Cocktail for General Use (MS-safe, 50X) , Protease Inhibitor Cocktail for Mammalian Cell and Tissue Extracts , Protease and Phosphatase Inhibitor Cocktail for Mammalian Cell and Tissue Extracts , etc.If using a vacuum pump to aspirate the supernatant, the strength of the vacuum pump should be controlled properly to avoid the aspiration of magnetic beads.Containing 0.1% non-ionic detergent (such as Triton X-100, Tween-20, and NP-40) in solutions can effectively prevent aggregation of magnetic beads, without affecting the antibody binding efficiency of the beads.High concentrations of DTT, mercaptoethanol, or guanidine hydrochloride, may have a certain effect on the binding of this product to MBP fusion proteins, but Cell lysis buffer for Western and IP , RIPA Lysis Buffer and NP-40 Lysis Buffer , are fully applicable. For the selection guide of different lysis buffers from , please refer to our website at: 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 samples:a. 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, RIPA Strong Lysis Buffer , RIPA Medium Lysis Buffer , or RIPA Weak Lysis Buffer can be attempted. 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-MBP Magnetic BeadsAs Anti-MBP magnetic beads are stored in a special protective solution, they need to be washed with an appropriate wash buffer before adding to samples.a. 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 the initial volume is greater than 0.2 ml, consider separating by placing directly on a magnetic rack (FMS012/FMS024) for 10 seconds to remove the supernatant before adding 1X TBS to a final volume of ~0.5 ml.b. Add 500μl of 1X TBS (, ST661/ST665) to the beads and pipette gently to mix. Place the tube into a magnetic stand for 10 seconds 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 mix well, and incubate for 2 hours at room temperature or overnight at 4℃ while gently rotating on a rotary mixer.Note: Occasional aggregation of magnetic beads during the binding process doesn't affect experimental results.b. Place the tube on a magnetic stand for 10s 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 on a magnetic stand for 10s to collect the beads against the side of the tube. Remove and discard the supernatant. Repeat this step 3 timesNote: 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 and downstream applications, one of the following two elution methods can be used.a. Elution with 1X SDS-PAGE loading buffer under denaturing conditions: The protein sample obtained is suitable for SDS-PAGE electrophoresis or Western blot.(a) Prepare SDS-PAGE loading buffe, or directly use 's SDS-PAGE protein loading buffer (1X) . Typically, SDS-PAGE protein loading buffer contains reducing agents such as DTT, and the eluted protein sample will contain the light and heavy chains of the MBP antibody.(b) Add 100μl of 1X SDS-PAGE loading buffer per 10-20μl of magnetic beads, and heat at 95℃ for 5 minutes.(c) Place the magnetic beads on a magnetic stand for 10 seconds and collect the supernatant for SDS-PAGE electrophoresis or Western blot analysis.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 activity analysis.(a) Prepare 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 rotary mixer for 5 minutes at room temperature (no more than 15 minutes). (c) Place the tube on 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 -80℃ for long-term storage. (e) To obtain higher elution efficiency, repeat steps b-c and combine the eluates.Note 1: Due to the strong binding affinity between the MBP tag and the MBP antibody, the effectiveness of acidic elution may be lower than that of SDS-PAGE loading buffer elution method.Note 2: The acidic elution efficiency varies depending 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, and the pH or amount of neutralization buffer needs to be adjusted correspondingly.FAQ: ProblemPossible CausesSolutionsVery few or no Flag‐tagged protein in the eluate.Protein is not completely elutedChange elution methods.No target protein expressedMake sure the target protein contains the MBP-tag by Western blot or dot blot analysis.Very low protein expression level1. Use larger volume of cell lysate.Optimize expression conditions to increase the protein expression level.Washes are too stringentRReduce the wash time and number of washes.Incubation time is inadequate.Prolong the incubation time.Interfering substances present in the sample.Lysates should not contain high concentration of DTT, 2-mercaptoethanol, or other reducing agents that may interfere with antibody-antigen interaction.Low sensitivity of the detection system.For Western blot analysis:1. Check the specificity and reactivity of primary and secondary antibodies using proper controls.2. Check the protein transfer efficiency by using 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 entire sample to a clean microcentrifuge tube before centrifugation.Insufficient washing of magnetic beads after protein binding1. 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 wash buffer.Centrifuge at lower speed to avoid nonspecific trapping of denatured proteins.Multiple protein bands found in the eluate.The protein is not stable at room temperaturePurify the target protein at lower temperature, such as 4℃.Protein degradation due to protease activity during the purification process.Add protease inhibitors to cell lysate.Non‐specific binding.1. Prepare cell lysate again.2. Add additional wash steps.References:1. Duplay P, Bedouelle H, Fowler A, Zabin I, Saurin W, et al. J Biol Chem. 1984. 259(16):10606-13.2. Reuten R, Nikodemus D, Oliveira MB, Patel TR, Brachvogel B, et al. PLoS One. 2016. 11(3):e0152386.
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