| Description | The HA tag consists of the amino acid sequence YPYDVPDYA (residues 98-106 of human influenza hemagglutinin). It has minimal impact on the tertiary structure of the target foreign protein and can be easily fused to either the N- or C-terminus, making it a popular choice for recombinant protein The HA tag consists of the amino acid sequence YPYDVPDYA (residues 98-106 of human influenza hemagglutinin). It has minimal impact on the tertiary structure of the target foreign protein and can be easily fused to either the N- or C-terminus, making it a popular choice for recombinant protein expression. Anti-HA Agarose Resin is based on a 4% agarose gel matrix, which minimizes non-specific binding of host cell proteins, making it suitable for both the purification and immunoprecipitation (IP) of HA-tagged fusion proteins.Aladdin Anti-HA Agarose Resin is stored in a solution containing 0.1% ProClin 300, with a settled gel to storage solution ratio of 1:1. The product specification refers to the actual volume of the settled gel.ParameterValue / DescriptionMatrix4% Agarose MicrospheresLigandAnti-HA Mouse Monoclonal AntibodyParticle Size Range45~165 µmBinding Capacity>1 mg HA-tagged protein / mL resinMaximum Pressure0.1 MPa, 1 barStorage Conditions0.1% ProClin 300, 2~8℃Shelf Life2 yearsProtocol1. Sample PreparationEnsure the sample solution has appropriate ionic strength and pH before loading. Dilute the sample or cell culture supernatant with equilibration buffer, or dialyze the sample against equilibration buffer.Clarify the sample by centrifugation or filtration through a 0.22 µm or 0.45 µm membrane to reduce impurities, improve purification efficiency, and prevent column clogging.2. Buffer PreparationIt is recommended to filter water and buffers through a 0.22 µm or 0.45 µm membrane before use.Equilibration Buffer: 10 mM Tris, 0.15 M NaCl, pH 7.4Wash Buffer: 10 mM Tris, 0.15 M NaCl, 0.05% Tween-20, pH 7.4Chemical Elution Buffers:0.1 M Glycine-HCl, pH 2.0–2.83 M Sodium Thiocyanate (NaSCN)50 mM NaOHCompetitive Elution Buffer: 50 mM Tris, 0.15 M NaCl, 100–500 µg HA peptide / mL, pH 7.4Neutralization Buffer: 1 M Tris-HCl, pH 8.5Comparison of Chemical Elution BuffersSolutionAdvantagesDisadvantages0.1M glycine HCl, pH 2.0-2.8Does not damage resin binding capacity if target protein is stable at low pHLow elution efficiency; target protein may denature3M NaSCNHigh elution efficiency; does not damage resin binding capacityTarget protein may denature50mM NaOHHigh elution efficiencyTarget protein may denature; reduces resin lifespan3. Sample Purification3.1 Column Chromatography(1) Pack the Anti-HA Agarose Resin into a suitable chromatography column. Equilibrate the column with 5 column volumes (CV) of Equilibration Buffer.(2) Load the sample onto the equilibrated resin. Collect the flow-through. The sample can be reloaded to increase binding efficiency.(3) Wash with 10–20 CV of Wash Buffer to remove non-specifically bound proteins. Collect the wash fractions.(4) Elution:* A. Acidic Elution: Elute with 5 CV of acidic elution buffer (e.g., 0.1 M Glycine-HCl, pH 2.0–2.8). Add a volume of Neutralization Buffer equal to one-tenth of the elution volume to each fraction to adjust the pH to 7.0–8.0. Collect fractions separately.* Note: After acidic elution, the resin must be immediately re-equilibrated. Do not expose the resin to the acidic elution buffer for more than 20 minutes.* B. Chemical Elution: Elute with 5 CV of a chemical elution buffer (e.g., 3 M NaSCN or 50 mM NaOH). Collect fractions separately.* Note: After chemical elution, the resin must be immediately re-equilibrated. Do not expose the resin to the chemical elution buffer for more than 20 minutes.* C. Competitive Elution: Elute with 5 CV of Competitive Elution Buffer. Collect fractions separately.(5) Regenerate the resin with 3 CV of a chemical elution buffer (e.g., Glycine-HCl), then re-equilibrate with Equilibration Buffer until neutral pH is reached.(6) Store the resin in Storage Buffer at 2–8°C.3.2 Batch/Binding Method(1) Resin Preparation: Transfer an appropriate amount of Anti-HA Agarose Resin to a column and drain the storage solution. Wash with 5 CV of Equilibration Buffer.(2) Add the sample solution. Incubate with shaking at 4°C or room temperature for 30 minutes (avoid magnetic stirring). Ensure thorough mixing.(3) After incubation, centrifuge the mixture (5,000 × g, 1 min) or filter to collect the resin.(4) Transfer the resin to a column. Wash with Equilibration Buffer until the UV baseline stabilizes.(5) Elute using either the Chemical or Competitive Elution method as described in section 3.1 (4).(6) Regenerate and store the resin as described in sections 3.1 (5) and (6).3.3 Immunoprecipitation (IP) Procedure(1) Resin Preparation: Add 40 µL of Anti-HA Agarose Resin suspension (20 µL settled resin) to a 1.5 mL tube. Centrifuge at 5,000 × g for 1 min. Discard the supernatant.(2) Add 0.5 mL of Equilibration Buffer to resuspend the resin. Centrifuge at 5,000 × g for 1 min. Discard the supernatant. Repeat this wash step once.(3) Add 200–1000 µL of sample lysate to the prepared resin. Mix and incubate on a tube rotator at room temperature for at least 1 hour. Centrifuge at 5,000 × g for 1 min. Collect the supernatant for analysis.(4) Washing: Add 0.5 mL of Wash Buffer, resuspend the resin, and mix gently. Centrifuge at 5,000 × g for 1 min. Discard the supernatant. Repeat this wash step three more times.(5) Elution: Choose the elution method based on downstream application.* A. Chemical Elution: Add 100 µL of chemical elution buffer (0.1 M Glycine-HCl pH 2.0-2.8, 3 M NaSCN, or 50 mM NaOH) and resuspend the resin. Incubate at room temperature for 5 min. Centrifuge at 5,000 × g for 1 min. Carefully collect the supernatant and neutralize immediately if acidic. Store eluted samples at 4°C short-term or -20°C long-term.* B. Competitive Elution: Add 100 µL of Competitive Elution Buffer and resuspend the resin. Incubate at room temperature for 30 min. Centrifuge at 5,000 × g for 1 min. Carefully collect the supernatant. Repeat elution 1-2 times. Store eluted samples at 4°C short-term or -20°C long-term.* C. Denaturing Elution (SDS-PAGE): Add 20 µL of 2× Loading Buffer (contains SDS and reducing agents like β-mercaptoethanol/DTT) to the resin. Heat at 95°C for 5 min. Centrifuge at 5,000 × g for 1 min, and load the supernatant directly onto an SDS-PAGE gel for analysis. Note: This method denatures the antibody, rendering the resin unusable for reuse.Troubleshooting Guide... Read More | Ganglioside GT1b is a brain ganglioside. It is composed of a neutral tetra-saccharide core, with one or two sialic acid on the internal galactose and an extra sialic acid on the non-reducing terminal of galactose | Laccase is an enzyme, produced by ericoid mycorrhiza and ectomycorrhiza fungi. It belongs to the group of polyphenol oxidases. Laccase is also present in plants and bacteria.Laccase from Trametes versicolor has been used: to assess the use of four laccase-producing strains in waste water treatment Laccase is an enzyme, produced by ericoid mycorrhiza and ectomycorrhiza fungi. It belongs to the group of polyphenol oxidases. Laccase is also present in plants and bacteria.Laccase from Trametes versicolor has been used: to assess the use of four laccase-producing strains in waste water treatment in laccase assay in screening the lignolsSome of the enzymatic actions of laccase are associated with sporulation, detoxification, morphogenesis, melanin polymerization and it offers protection to spore coat. Laccase can catalyse a number of substrates including medicinal drugs and halogenated pesticides. It utilizes oxygen for its catalysis. For these reasons, it might be useful in the biological degradation of micropollutants in wastewater treatment. Laccase catalyzes the oxidation of phenol containing compounds, including lignin, through the reduction of oxygen to water. The presence of mediators will allow the oxidation of non-phenlic compounds as well. The primary function of laccase is to degrade lignin in fungi... Read More | Inquire | Trypsin is a pancreatic serine protease with substrate specificity based upon positively charged lysine and arginine side chains. It is derived from a 34 kDa inactive precursor zymogen, trypsinogen, after enzymatic removal of an N-terminal 6-amino acid leader sequence resulting in the 23.8 kDa Trypsin is a pancreatic serine protease with substrate specificity based upon positively charged lysine and arginine side chains. It is derived from a 34 kDa inactive precursor zymogen, trypsinogen, after enzymatic removal of an N-terminal 6-amino acid leader sequence resulting in the 23.8 kDa trypsin molecule. The optimum pH is 8.0. Trypsin is inhibited by organophosphorus compounds such as diisopropylfluorophosphate and natural inhibitors from pancreas. Soybean, lima bean, and egg white are also sources of natural inhibitors. Trypsin cleaves amide and ester bonds of Arg and Lys. The Aladdin Sequencing Grade Trypsin has been further purified to remove trace contaminating proteases and autolysis products which could interfere in trypsin digestion experiments, and exhibits a single band on PAGE.Trypsin is a serine protease used to hydrolyze proteins. Trypsin from bovine pancreas has a molecular weight of 23.8 kDa. Trypsins are used for the re-suspension of cells during cell culture and in proteomics research for the digestion of various proteins... Read More |