| Product Description | Aladdin's UltraBio™ GST-tag Protein Purification Magnetic Agarose Beads are prepared by covalently coupling high-quality reduced L-Glutathione to agarose beads. This product can selectively bind GST fusion proteins in lysates, serum, ascites, or other samples from animals, plants, or microorganisms, and thus can be used for purification, immunoprecipitation (IP), or co-immunoprecipitation (Co-IP) of GST-tagged proteins or their protein complexe. The beads are compatible with 6M guanidine hydrochloride or 8M urea.GST (glutathione S-transferase) plays an important role in the detoxification process, with a molecular weight of approximately 26kDa. It can be fused to the N-terminus or C-terminus of a target protein for convenient purification and detection. There are several advantages for using the GST as a tag. It can act as a chaperone to facilitate protein folding and increases the solubility of GST fusion proteins. It can be expressed in different hosts such as E. coli and yeast, and has a wide range of applications. It can well retain the antigenicity and biological activity of target proteins, which helps protect the recombinant protein from degradation by extracellular proteases. The GST tag can be easily removed if there is a site-specific protease recognition site between the GST tag and the target protein, such as PreScission Protease, TEV Protease, or Thrombin. The GST fusion protein can be purified specifically and conveniently by using Aladdin's GST Antibody (AH158), Anti-GST Magnetic Beads, UltraBio™ GST-tag Purification Resin, or GST Tag Protein Purification Kit . The fusion protein can also be easily detected, characterized, and used for IP or Co-IP analysis. Therefore, GST tags have been widely used in protein expression, purification, identification, protein-protein interaction, and functional studies.UltraBio™ GST-tag Protein Purification Agarose Magnetic Beads specifically bind GST fusion proteins. With a magnetic stand, this product can be conveniently used for protein purification and immunoassays. Please refer to Figure 1 for the workflow of immunoassays with this product.Figure 1. The workflow of immunoassays using the UltraBio™ GST-tag Protein Purification Magnetic Agarose Beads .This product is highly stable, with strong specificity and high binding capacity for GST fusion proteins. Compared to most other similar products, this product has a high density of reduced L-glutathione ligands that bind GST fusion proteins with strong specificity. Each milliliter of suspension contains approximately 0.25ml settled magnetic agarose beads and no less than 30-50μmol/ml of GSH. Typically, each milliliter of beads can bind 10-20mg of GST fusion protein, depending on the size of fusion proteins.This product can bind GST-tagged proteins of various forms, such as N-terminal methionine GST fusion proteins (Met-GST-Protein), N-terminal GST fusion proteins , and C-terminal GST fusion proteins (Protein-GST).This product binds GST-tagged proteins rapidly. This product has a particle size of approximately 100μm. It usually takes 30 minutes for the binding and 60 minutes to complete the purification or immunoprecipitation of GST fusion proteins, during which protein degradation and denaturation can be minimized effectively.This product is compatible with multiple elution methods. The MBP-tagged proteins can be eluted using the 1X SDS-PAGE loading buffer or acidic elution buffers, depending on the structure, biological function, and downstream applications of the target protein. Please refer to Figure 2 for the IP result using this product.Figure 2. SDS-PAGE analysis of GST-tagged proteins purified using the UltraBio™ GST-tag Protein Purification Magnetic Agarose Beads . 1, Total lysate of E. coli expressing GST-tagged proteins; 2&3, First and second eluates from the UltraBio™ GST-tag Protein Purification Magnetic Agarose Beads; 4&5, First and second eluates from Competitor G Magnetic Agarose Beads. This figure is for reference only, which may vary due to different experimental conditions.This product is convenient to use. It is stored in a special protective solution without glycerol and can be quickly and efficiently separated from the solution by using a magnetic stand, eliminating the need for centrifugation.Main characteristics of this product
Precautions： This product can tolerate 6M guanidine hydrochloride or 8M urea. However, the purification of GST-tagged proteins should always be performed under non-denaturing conditions. If fusion proteins in inclusion bodies have to be dissolved in 8M urea or 6M guanidine hydrochloride, the urea or guanidine hydrochloride should be removed by dialysis and the protein should be refolded before using this product for purification.The product should be maintained at a pH of 6-8. Do not centrifuge or dry the magnetic beads. Long time exposure of the beads to magnetic field will cause beads to agglomerate.Resuspend the magnetic agarose beads thoroughly in solution by gentle pipetting prior to use. Vigorous vortex or shaking should be avoided.For purification or immunoprecipitation, it is recommended to set up both positive and negative controls appropriately.The GSH in elution buffer is prone to oxidation, so it is recommended to prepare and use it fresh.Including 1-10mM DTT in the sample or buffer may promote the binding of some GST fusion proteins to the magnetic agarose beads.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 GST 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： This protocol is described for the purification of GST-tagged recombinant proteins expressed in Escherichia coli. Typically, each 500ml of E. coli culture yields 2-4g wet weight of bacterial cells. Use 4ml of magnetic agarose bead suspension for protein purification, and the protein yield is expected to be 10-20mg. The scale of the experiment can be adjusted as needed.1. Preparation of buffersBufferComponentsBinding/Wash Buffer1X PBS, pH7.4Elution Buffer50mM Tris-HCl, 10mM GSH, pH8.0Note: It is recommended to filter the solutions with 0.22μm or 0.45μm filter membranes before use to improve the protein purification efficiency of this product.2. Preparation of protein samplesa. Centrifuge the E. coli culture at 4℃ for 20 minutes at 4,000×g or 1 minute at 15,000×g to collect cell pellet, and discard the supernatant. Proceed to bacterial lysis or store the pellet at -20℃ or -80℃ for later use. Thaw the frozen cells on ice for 15 minutes before use.b. Resuspend the bacterial pellet thoroughly in Binding/Wash Buffer. If necessary, add an appropriate amount of protease inhibitor cocktail, such as Super Nuclease (≥99%) ( GST-tag Protein Purification Magnetic Agarose Beadsa. Gently resuspend the magnetic agarose beads by pipetting and transfer an appropriate amount to a new centrifuge tube ( Digital See-Saw Rocking Shaker . b. After incubation, place the mixture on a magnetic rack for 10 seconds, remove and discard the supernatant.c. Wash beads with 2ml of Binding/Wash Buffer by gentle pipetting to resuspend the beads completely. Place the tube on a magnetic rack for 10 seconds, remove and discard the supernatant. Repeat this step thrice.5. Elutiona. Depending on the concentration of the target protein and the requirements of downstream applications, add 0.2-1ml of Elution Buffer. Gently flip the centrifuge tube a few times to suspend the magnetic agarose beads and incubate for 5 minutes. Place the tube on a magnetic rack for 10 seconds and collect the supernatant containing the purified GST-tagged proteins into a new centrifuge tube.b. If necessary, perform a second elution to improve the yield.6. Cleaning and regeneration of magnetic agarose beadsa. After protein elution, add 2ml of Elution Buffer to the IDA-Ni beads and resuspend the beads by inverting the tube several times. Vortex for 10 seconds, place the tube on a magnetic rack for 10 seconds, remove and discard the supernatant. Repeat this step thrice.b. Add 2ml of 20% ethanol and resuspend the beads by inverting the tube several times. Vortex for 10 seconds, place the tube on a magnetic rack for 10 seconds, remove and discard the supernatant. Repeat this step thrice.c. Store the magnetic beads in 20% ethanol, making the total volume equal to the initial bead suspension volume. Store at 2-30℃ (for long-term storage, store at 2-8℃). The beads can be reused to purify the same protein.ProblemsPossible CausesSolutionsVery few or no GST-tagged protein exists in the eluateProtein is not completely eluted.1. Increase the GSH concentration to 15mM or higher in the Elution Buffer.2. Increase the elution or incubation time.Add Triton X-100 (0.1%) or n-Octylglucoside (2%) or NaCl (0.1-0.2M) to the Elution Buffer.No fusion protein expressed.Make sure the target protein has the GST-tag by Western blot or dot blot analysis.Very low protein expression level.1. Use larger volume of cell lysate.2. Optimize expression conditions to increase the protein expression level.Washes are too stringent.Reduce the wash time and number of washes.Incubation times are inadequate.Increase the incubation time.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 purity of the target protein is lowThe target protein has degraded.Add appropriate protease inhibitors such as PMSF to the cell lysate and the Binding/Wash Buffers.Host proteins, such as chaperonins, may interact with the fusion protein.1. Add DTT (5mM) in Binding/Wash Buffers.2. Add Chaperonin Buffer (2mM ATP, 10mM MgSO4, 50mM Tris-HCl) to the cell lysate and incubate at 37℃ for 10 minutes prior to the purification.Insufficient washing of beads after protein binding.1. Increase the number of washes.2. Prolong duration of each wash to at least 15 minutes.3. Use more stringent wash conditions. Detergent such as 1% Triton X-100, 1% Tween-20, 0.03% SDS, or 0.1% NP-40 can be attempted. 4. Centrifuge at lower speed to avoid nonspecific trapping of denatured proteins.The binding capacity of the magnetic beads has declinedAggregation of protein or other substances on the beads.Wash the beads with NaOH.The beads have been reused too many times.Use new beads.References:1. Einarson MB, Pugacheva EN, Orlinick JR. CSH Protoc. 2007. 2007:pdb.prot4757.2. Schäfer F, Seip N, Maertens B, Block H, Kubicek J. Methods Enzymol. 2015. 559:127-39.
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