| 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 | Inquire | Product introduction：Aladdin ® SE is a kind of fluorescent dye with amino reactive activity. The SE group of these dyes can react with the amino group to produce a stable amide bond. Compared with other similar dyes on the market, aladdin ® is a new generation of fluorescent dyes Product introduction：Aladdin ® SE is a kind of fluorescent dye with amino reactive activity. The SE group of these dyes can react with the amino group to produce a stable amide bond. Compared with other similar dyes on the market, aladdin ® is a new generation of fluorescent dyes with stronger stability, better water solubility and better fluorescence intensity. Product parameters： Absmax/Em(nm)：648/664；Absmax/Em(nm)：0.03；Extinction coefficient(ε)：240000；Optimal DOL(IgG)：3-6； Usage:1. Experimental materials(1) IgG: IgG must not contain amine chemicals that can react with dyes, such as amino acids, Tris, BSA, gelatin, etc. If IgG contains such chemicals, PBS buffer with pH~7.4 should be used for pre dialysis treatment. The presence of azide compounds does not affect the labeling reaction.(2) Anhydrous DMSO(3) NaHCO3(4) Sephadex gel G-25 dialysis column(5) PBS buffer (pH~7.4)(6) NaN3(7) BSA2. Marking methods and steps(1) Prepare to label antibodiesDilute the antibody with 0.1 M NaHCO3 solution (pH~8.3) to a final concentration of 2.5 mg/mL. If the product is pre diluted with phosphate buffer, such as PBS buffer (without amino compounds), approximately 1/10 volume of 1M NaHCO3 mother liquor can be directly added to the buffer to achieve a final NaHCO3 concentration of 0.1 M.Note: When the protein concentration is 2.5 mg/mL, the labeling efficiency is approximately 35%. Protein concentrations below 2.5 mg/mL can also be used for labeling, but the labeling efficiency will decrease. When the protein concentration is higher than 5 mg/mL, the labeling efficiency may be higher. Due to differences in buffer and protein purity, more precise labeling efficiency is determined by practical operating conditions. If the protein concentration is too low, it can be concentrated by ultrafiltration.(2) Prepare dye storage solutionPreheat one tube at room temperature µ YF of Mole ® SE, add 0.1 mL of anhydrous DMSO to the tube, thoroughly vortex dissolve the dye, and prepare a dye storage solution with a concentration of 10 mM. If a trace amount of protein is used for labeling reactions, the dye needs to be diluted to a lower concentration.Note: a The remaining dye storage solution should be stored at a low temperature of -20 ℃ for future use. If anhydrous DMSO is used to prepare dye storage solution, the dye can be stored for at least one month.b. Dyes can also be prepared with deionized water, but due to the slow hydrolysis of dyes in water, it is best to prepare water based storage solutions for immediate use.(3) Mark reaction stepsa. Stir or vortex the protein solution, gradually adding 15-25 drops µ L dye storage solution (10 mM), with a molar ratio of dye/protein in the range of 9:1 to 15:1. YF ® Please refer to the table above for the range of DOL (number of dyes bound to each protein molecule) for SE labeled IgG antibodies.b. Stir the reaction at room temperature for 1 hour, and for trace labeling, shake and incubate on a shaker for 1 hour.Note: At the same time of the binding reaction, proceed to step 2 (4) to balance the dextran gel G-25 dialysis column.(4) Isolation of marker proteins from reaction solutiona. PBS buffer (pH~7.4) was used to balance the dextran gel G-25 dialysis column (10 mm × 300 mm).b. Add the reaction solution from step 3 (b) to the column and elute with 1 x PBS buffer.The first washed out chromophore is a dye protein complex.Note: a For small-scale labeling reactions, in order to avoid excessive dilution of the product, ultrafiltration devices can be used to remove free dyes from the complex.b. After the binding reaction is completed, if the dye protein complex is not separated in time, 50 can be added µ Terminate the reaction with L 1M lysine. In most cases, this operation is not necessary because the remaining unreacted dyes have been fully hydrolyzed at the end of the reaction.3. Determine DOL(1) The determination of protein concentration and antibody concentration can be calculated using the following formula:C (mg/mL)={[A280- (Amax x x Cf)]/1.4} x dilution factor;a. C refers to the concentration of antibodies collected in the experiment;b. Dilution factor refers to the dilution factor used in photometric measurements;c. A280 and Amax refer to the absorbance at 280 nm and the absorbance at the absorption wavelength, respectively;d. Cf is the correction factor, YF ® Please refer to the table above for the Cf value of SE dyes;Note: The protein solution eluted through the column may have a high concentration when used directly for absorbance detection, so it needs to be diluted to approximately 0.1 mg/mL. The dilution factor (i.e. dilution factor) needs to be determined from the initial number of antibodies (e.g. 5 mg) and the overall elution of protein solutionEstimate based on the product.(2) Estimation of DOLDOL is calculated using the following equation:DOL=(Amax x x Mwt x Dilution Factor)/（ ε X C)a. Amax, dilution factor, C value has been clearly defined in 3 (1);b. Mwt refers to the molecular weight of IgG (150000);C. c ε It's YF ® The molar absorption coefficient of SE, refer to the table on the first page;d. Mark YF ® The optimal DOL value for SE IgG antibodies can be found in the table on the first page. Although DOL values may fluctuate, good experimental results can also be achieved.Matters needing attention：1. if the labeled protein needs long-term storage, it is recommended to add 5-10 mg/ml BSA and 0.01-0.03% NaN3 to prevent protein denaturation and microbial breeding. Store at 4 ℃ away from light. If glycerol with a final concentration of 50% is added, it can be stored at -20 ℃. It can be stably stored for more than one year. 2. keep away from light during operation. The mixing speed should be appropriate to avoid bubbles. 3. when installing the chromatographic column, try to make the column body uniform, the column surface flat, and free of bubbles and cracks. 4. pay attention to adding the sample when the column top buffer is tangent to the gel plane. When eluting, add the eluent when the sample is tangent to the gel plane. 5. other factors affecting the labeling efficiency also include temperature, reaction time, pH, the amount of fluorescent dye and protein, etc., which should be controlled. 6. for your safety and health, please wear laboratory clothes and disposable gloves.Scope of application：Protein nucleic acid labeling dye... Read More | Store at +4°C. Store under desiccating conditions. The product can be stored for up to 12 months | Purity> 96% by SDS-PAGE and HPLC analyses.FunctionHas weak activities on human monocytes and acts via receptors that also recognize MIP-1 alpha. It induced intracellular Ca(2+) changes and enzyme release, but no chemotaxis, at concentrations of 100-1,000 nM, and was inactive on T-lymphocytes, Purity> 96% by SDS-PAGE and HPLC analyses.FunctionHas weak activities on human monocytes and acts via receptors that also recognize MIP-1 alpha. It induced intracellular Ca(2+) changes and enzyme release, but no chemotaxis, at concentrations of 100-1,000 nM, and was inactive on T-lymphocytes, neutrophils, and eosinophil leukocytes. Enhances the proliferation of CD34 myeloid progenitor cells. The processed form HCC-1(9-74) is a chemotactic factor that attracts monocytes eosinophils, and T-cells and is a ligand for CCR1, CCR3 and CCR5.Post-translationalThe N-terminal processed forms HCC-1(3-74), HCC-1(4-74) and HCC-1(9-74) are produced in small amounts by proteolytic cleavage after secretion in blood. HCC-1(1-74), but not HCC-1(3-74) and HCC-1(4-74), is partially O-glycosylated; the O-linked glycan consists of one Gal-GalNAc disaccharide, further modified by two N-acetylneuraminic acids... Read More |