| Product Description | Aladdin's UltraBio™ Anti-GFP Magnetic Beads are based on nano-scale magnetic beads covalently coupled with high-quality GFP mouse monoclonal antibodies that specifically bind to GFP-tagged proteins in animal, plant or microbial lysate, serum, ascites, etc. This product is mainly used for immunoprecipitation (IP), co-immunoprecipitation (Co-IP) or purification of GFP-tagged fusion proteins or protein complexes.GFP (green fluorescent protein) was first discovered by Osamu Shimomura, Qian Yongjian and others in Aequorea victoria in 1962. When excited by ultraviolet or blue light, it emits green fluorescence. The fusion expression of GFP or its mutant EGFP (enhanced green fluorescent protein) and the target protein is widely used for gene expression regulation, transgene function, expression, distribution and migration of target protein in cells, high-throughput drug screening, etc. The advantage of the GFP label is that it is easy and suitable for real-time observation of the localization and expression of the target protein in the cell without the need of sample lysis and substrate or antibody additions. Moreover, the fluorescence of GFP is stable and other components in the cell generally do not interfere with the detection of GFP-tagged protein, so that the detection is fast, simple, sensitive, and reproducible. However, the molecular weight of the GFP tag is relatively large , which may affect the function of the target protein when fused with GFP.Store at 4℃ for up to 2 years, or at -20℃ for long term storage.UltraBio™ Anti-GFP Magnetic Beads specifically binds with proteins tagged with GFP or EGFP. With magnetic stand, this product can be conveniently used for immunoprecipitation or purification of fusion proteins or protein complexes with GFP tags. Please refer to Figure 1 for the IP assays with this product. Figure 1. The work flow of IP assays with the UltraBio™ Anti-GFP Magnetic Beads .This product has strong specificity and high binding capacity of target protein. Compared with most similar products, this product has higher binding capacity and specificity for GFP-tagged proteins, and the small particle size of magnetic beads makes it less prone to non-specific adsorption. Each milliliter of the beads suspension contains about 10mg magnetic beads coupled with no less than 0.6mg Anti-GFP antibody. It can bind no less than 0.6mg GFP-tagged proteins, which is usually dependent on the molecular weight of the tagged protein. Immunoprecipitation can be performed with 10-20μl of bead suspension for every 500μl of cell lysate.This product can bind to various forms of GFP-tagged protein. This product can specifically bind to both N-terminal GFP fusion protein (GFP-Protein) and C-terminal GFP fusion protein (Protein-GFP).This product binds to GFP-tagged proteins rapidlyt. This product uses nano-scale magnetic beads (~200nm) with a large specific surface area, enabling a fast and efficient binding between antibody and antigen. It usually takes 10 minutes for the binding and 30 minutes to complete the whole process of immunoprecitation, during which protein degradation and denaturation can be minimized effectively.This product is compatible with a variety of elution methods. The GFP-tagged protein can be eluted using SDS-PAGE loading buffer and acidic eluents, depending on the structure of the target protein, its biological function, and the requirements of subsequent applications. Please refer to Figure 2 for the IP result using this product. Figure 2. The immunoprecipitation effect of UltraBio™ Anti-GFP Magnetic Beads for GFP-tagged protein. 293T (human embryonic kidney) cells were transfected with the plasmid carrying the gene encoding GFP-tagged protein. After 36 hours post infection, cells were lysed using the Cell lysis buffer for Western and IP and immunoprecipation was performed with the cell lysate. Lane 1, Total cell lysate (Input); Lane 2, Mouse IgG Magnetic Beads negative control; Lane 3, IP results with the cell lysate and the UltraBio™ Anti-GFP Magnetic Beads , eluted with SDS-PAGE protein loading buffer (1X). The western membrane was blotted with anti-GFP antidody and examined by UltraBio™ 600 chemiluminescence imaging system . This figure is for reference only, which may vary due to different experimental conditions.This product is easy to use. This product is stored in a special protective solution free from glycerol, and can be quickly and efficiently separated from the solution by using a magnetic stand.Main parameters of the UltraBio™ Anti-GFP Magnetic Beads
Precautions： This product should be maintained at pH 6-8. Do not centrifuge, dry or freeze the magnetic beads. Long time exposure of the beads to magnetic field will cause beads to agglomerate.Resuspend Mag™ Anti-GFP Magnetic Beads evenly in solution by gentle pipetting prior to use. Do not vortex to avoid the denaturation of antibodies.When performing precipitation or purification, it is recommended to set up both positive and negative controls appropriately.The 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 ’s 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.Magnetic beads may aggregate when eluted with acidic solutions, which is a normal phenomenon and does not affect the normal use of magnetic beads. 0.1% non-ionic detergent (such as Triton X-100, Tween-20 or NP-40) can effectively prevent magnetic beads from agglomerating without affecting the antibody binding efficiency of magnetic beads.High concentration of DTT, mercaptoethanol, guanidine hydrochloride, etc. may have a certain effect on the binding of this product to the tagged protein, but Cell lysis buffer for Western and IP , RIPA Lysis Buffer or NP-40 Lysis Buffer , etc. are completely applicable. The main features and differences of the different lysis solutions produced by , and how to choose the lysis solution can refer to our related 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. Sample preparationa. Lysis cells or tissues with appropriate lysis buffer. We recommend using the Cell Lysis Buffer for Western and IP in priority. Homemade or other company's lysis buffer with a pH of 6-8 can also be used.Note: The large molecular weight of GFP tag itself and the structural complexity of the fusion protein may affect the recognition of the N-terminal of the GFP protein by the Anti-GFP magnetic beads, so we recommend verifying the immunoprecipitation or adjusting the lysis conditions through preliminary experiments.b. Prepare the cell or tissue lysate according to the instructions of the lysis buffer. After lysis and centrifugation, keep the supernatant on ice or at 4ºC for subsequent use. We recommend performing subsequent procedures such as immunoprecipitation on the same day as the protein sample is prepared. Otherwise, make aliquot and store at -80ºC for future use.2. Preparation of Anti-GFP magnetic beadsSince Anti-GFP magnetic beads are stored in a special protective solution, they need to be washed properly 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 protein sample.Note: If using more than 0.2 ml of bead suspension, place the tube into a magnetic stand (FMS012/FMS024) to collect beads and remove the supernatant.b. Add 500μl of 1X TBS 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 for 2 times.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 beads suspension is taken, add 10μl of 1X TBS).3. Immunoprecipitation (IP)a. Incubation: 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: Magnetic beads may aggregate during incubation, which is a normal phenomenon and does not affect the result.b. Magnetic separation: Place the tube on a magnetic stand for 10 seconds to collect the beads against the side of the tube. Remove and discard the supernatant. Note: A small amount of supernatant can be reserved in a clean centrifuge tube for examination of the binding results.c. Wash: Add 500μl of 1X TBS to resuspend the beads by gently pipetting. Place the tube on a magnetic stand for 10 seconds to collect the beads and remove the supernatant. Repeat the wash 3 times. Note: The A280 of supernatant can be measured to determine whether the beads are washed thoroughly. Repeat wash until the A280 is smaller than 0.05.4. ElutionAccording to the characteristics of the tagged protein and subsequent experimental requirements, one of the following two methods can be selected for elution.a. Elution with 1X SDS-PAGE loading buffer under denaturing conditions. The eluate can be used for gel electrophoresis and immuoblotting. .(a)Preparation of SDS-PAGE loading buffer:The SDS-PAGE loading buffer can be homemade or purchased from Protein A Magnetic Beads1mlP2102-5ml Protein A Magnetic Beads5mlP2105-1ml Protein G Magnetic Beads1mlP2105-5ml Protein G Magnetic Beads5mlP2108-1ml Protein A+G Magnetic Beads1mlP2108-5ml Protein A+G Magnetic Beads5mlP2115-0.5ml Anti-Flag Magnetic Beads0.5mlP2115-2ml Anti-Flag Magnetic Beads2mlP2118-0.5ml Anti-Myc Magnetic Beads0.5mlP2118-10ml Anti-Myc Magnetic Beads10mlP2118-2ml Anti-Myc Magnetic Beads2mlP2121-0.5ml Anti-HA Magnetic Beads0.5mlP2135-0.5ml Anti-His Magnetic Beads0.5mlP2135-2ml Anti-His Magnetic Beads2mlP2138-0.5ml Anti-GST Magnetic Beads0.5mlP2138-2ml Anti-GST Magnetic Beads2mlP2141-0.5ml Anti-V5 Magnetic Beads0.5mlP2141-2ml Anti-V5 Magnetic Beads2mlP2151-1ml Streptavidin Magnetic Beads1mlP2151-200μl Streptavidin Magnetic Beads200μlP2151-5ml Streptavidin Magnetic Beads5mlP2171-1ml Mouse IgG Magnetic Beads1mlP2171-5ml Mouse IgG Magnetic Beads5mlP2173-1ml Rabbit IgG Magnetic Beads1mlP2173-5ml Rabbit IgG Magnetic Beads5ml
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