| 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 | 1、Product attributeReaction time：short (up to 20 minutes) at 20-37°CLot-to-lot variation：<5%Boiling point : 100℃pH-Value (at 20 °C): 3.5-4.0Density (20℃) : 1.0111 g/cm³Appearance: colourless to pale blue liquidOdour: odourlessRecommend Incubation 1、Product attributeReaction time：short (up to 20 minutes) at 20-37°CLot-to-lot variation：<5%Boiling point : 100℃pH-Value (at 20 °C): 3.5-4.0Density (20℃) : 1.0111 g/cm³Appearance: colourless to pale blue liquidOdour: odourlessRecommend Incubation temperature: 20-37 °C2、Requirements for storage rooms and vessels1.Keep container tightly closed.2.Keep cool. protected from light3.Keep/Store only in original container.4.Never return spills in original containers for reuse.5. Keep away from: Food and feeding stuffs3、It is a ready-to-use, labelling-free TMB-substrate solution.4、Biosafety informationThis mixture is not classified as hazardous in accordance with Regulation (EC) No 1272/2008；5、Advantage1. Very high absorbance yield2. Very low background signals3. Certified long-term stability4. Regeneration following light exposure... Read More | Product contentG665787Component5 mLStorageG665787A2×GoldStar Probe Mixture (UNG)5×1 mL-20℃. Avoid freeze/thaw cycle.G665787B50×High ROX200 µL-20℃. Avoid freeze/thaw cycle.G665787CddH2O 5×1 mL -20℃. Avoid freeze/thaw cycle. Product Introduction2× Product contentG665787Component5 mLStorageG665787A2×GoldStar Probe Mixture (UNG)5×1 mL-20℃. Avoid freeze/thaw cycle.G665787B50×High ROX200 µL-20℃. Avoid freeze/thaw cycle.G665787CddH2O 5×1 mL -20℃. Avoid freeze/thaw cycle. Product Introduction2× GoldStar Probe Mixture (UNG) is a premixed system dedicated to real-time fluorescence quantitative PCR by probe method (TaqMan, Molecular Beacon, etc.), with a concentration of 2×, containing GoldStar Taq DNA polymerase, PCR Buffer, dNTPs (dTTP is all replaced by dUTP), UNG enzyme and Mg2+, which is easy and convenient to operate. It is mainly used for the detection of genomic DNA target sequences and cDNA target sequences after RNA reverse transcription, such as gene expression analysis, copy number analysis and SNP genotype analysis. This product utilizes the dUTP-UNG anti-pollution system, which adds dUTP during the preparation of the PCR reaction system, thus forming an amplification product containing dU bases. This product can be eliminated by the UNG enzyme in the PCR system before the next PCR reaction. This effectively removes residual contamination of the PCR product and greatly reduces false positives due to contamination of the amplification product.UNG enzyme can be inactivated at the pre-denaturation step in the PCR cycle, and therefore will not affect the formation of new PCR products containing dU bases. The GoldStar Taq DNA Polymerase contained in this product is a chemically modified, new high-efficiency hot-start enzyme, which has no polymerase activity at room temperature, effectively avoiding non-specific amplification due to non-specific binding of primers and templates or primer dimerization at room temperature, and the activation of the enzyme must be incubated at 95°C for 10 minutes. The unique combination of PCR buffer system and hot-start enzyme significantly improves the amplification efficiency of PCR with stronger fluorescent signal and higher sensitivity to detect single-copy templates. A wider linear range and more accurate quantification of the target gene can be obtained by using this product.ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used in Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument.Instruments that do not require ROX calibration (G670150):Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others.Instruments requiring Low ROX calibration(G665780):ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA 7 system. Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and more.Instruments requiring High ROX calibration(G665787):ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.matters needing attentionBefore use, please mix gently by turning up and down, avoid foaming as much as possible, and use after brief centrifugation.Avoid repeated freezing and thawing of this product, repeated freezing and thawing may degrade the product performance. This product can be stored for long term at -20℃, protected from light. If frequent use is required within a short period of time, it can be stored at 2-8℃.UsageThe following examples are conventional PCR reaction systems and reaction conditions, which should be improved and optimized according to the template, primer structure and target fragment size in actual operation.1.PCR reaction systemReagents50 µl Reaction systemfinal concentration2×GoldStar Probe Mixture（UNG）25 µl1×Forward Primer，10 µM1 µl0.2 µM¹⁾Reverse Primer，10 µM1 µl0.2 µM¹⁾Probe，10 µM1 µl0.2 µM²⁾Template DNA2 µl³⁾ 50×Low ROX or High ROX（optional）⁴⁾1 µl1×ddH₂Oup to 50 µlNote: 1) Usually, better results can be obtained with a primer concentration of 0.2 µM, and 0.1-1.0 µM can be used as a reference for setting the range.(2) The concentration of the probe used is related to the fluorescence quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, please refer to the instrument manual or the specific requirements for the use of each fluorescent probe for the adjustment of the concentration in actual use.(3) Usually the amount of DNA template is 10-100ng genomic DNA or 1-10ng cDNA as a reference. Since the templates of different species contain different copy numbers of target genes, the templates can be subjected to gradient dilution to determine the optimal amount of template to be used.(4) The excitation optical system varies from instrument to instrument, choose to add 50×Low ROX or 50×High ROX according to the instrument using fluorescence quantification.2.PCR reaction programCaution! The pre-denaturation reaction of this product must be completed at 95°C for 10 minutes!Two-step PCR:Note: 1) The hot-start enzyme used in this product must be activated under the condition of pre-denaturation 95℃, 10min. 2) It is recommended to use two-step PCR reaction program, if you can't get good experimental results due to the use of primers with lower Tm value, etc., you can try to carry out three-step PCR amplification.Three-step PCR:... Read More | Inquire | Tyrosine decarboxylase catalyzes the removal of the carboxyl group from tyrosine to produce tyramine and carbon dioxide. Pyridoxal 5'-phosphate is a necessary cofactor. By using the apoenzyme prepared from cells grown on a vitamin B6 deficient medium pyridoxal phosphate may be determined. The Tyrosine decarboxylase catalyzes the removal of the carboxyl group from tyrosine to produce tyramine and carbon dioxide. Pyridoxal 5'-phosphate is a necessary cofactor. By using the apoenzyme prepared from cells grown on a vitamin B6 deficient medium pyridoxal phosphate may be determined. The HOLOenzyme may be used to determine tyrosine, phenylalanine and dihydroxyphenylalanine either manometrically or colorimetrically.L-Tyrosine decarboxylase apoenzyme from Streptococcus faecalis has been used in a study to purify and characterize tyrosine decarboxylase and aromatic-L-amino-acid decarboxylase.L-Tyrosine decarboxylase apoenzyme from Streptococcus faecalis has also been used in a study to investigate the stereospecificity of sodium borohydride reduction of tyrosine decarboxylase.One Unit yields 1µmole of CO2 per minute from L-tyrosine at 37°C, pH 5.5. The APOenzyme activity is measured in the presence of excess pyridoxal phosphate... Read More |