| Description | The Flag tag is an octapeptide composed of hydrophilic amino acids, strategically positioned on the surface of fusion proteins. This location facilitates easier binding to antibodies and cleavage by enterokinase. Anti-Flag Agarose Resin utilizes an anti-Flag (DYKDDDDK) antibody as the affinity The Flag tag is an octapeptide composed of hydrophilic amino acids, strategically positioned on the surface of fusion proteins. This location facilitates easier binding to antibodies and cleavage by enterokinase. Anti-Flag Agarose Resin utilizes an anti-Flag (DYKDDDDK) antibody as the affinity ligand for the one-step purification of Flag-tagged fusion proteins expressed in prokaryotic, yeast, or mammalian cell systems. This product 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 Flag-tagged fusion proteins. Aladdin Anti-Flag 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-DYKDDDDK AntibodyParticle Size Range45~165 µmBinding Capacity>1 mg DYKDDDDK-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/Wash Buffer: 50 mM Tris, 0.15 M NaCl, pH 7.4Acidic Elution Buffer: 0.1 M Glycine-HCl, pH 3.0Competitive Elution Buffer: 50 mM Tris, 0.15 M NaCl, 100-500 µg Flag peptide / mL, pH 7.4Neutralization Buffer: 1 M Tris-HCl, pH 8.03. Sample Purification3.1 Column Chromatography(1) Pack the Anti-Flag Agarose Resin into a suitable chromatography column. Equilibrate the column with 5 column volumes (CV) of Equilibration Buffer to bring the resin into the same buffer system as the target protein.(2) Load the sample onto the equilibrated Anti-Flag Agarose Resin. Collect the flow-through. The sample can be reloaded to increase binding efficiency.(3) Wash with 10 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. 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 Anti-Flag Agarose Resin to the acidic elution buffer for more than 20 minutes.*** B. Competitive Elution: Elute with 5 CV of Competitive Elution Buffer. Collect fractions separately.(5) Regenerate the resin with 3 CV of the respective Elution Buffer, 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-Flag 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 at least 30 minutes (avoid magnetic stirring). Ensure thorough mixing of the resin and sample.(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 Acidic 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-Flag Agarose Resin suspension (20 µL settled resin) to a 2 mL tube. Centrifuge at 5,000 × g for 1 min. Carefully remove and discard the supernatant.(2) Add 0.5 mL of Equilibration Buffer to resuspend the resin (this brings it into the correct buffer system, protecting the protein). 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 thoroughly and incubate on a tube rotator or roller mixer at room temperature for at least 1 hour to facilitate binding. Centrifuge at 5,000 × g for 1 min. Discard the supernatant.(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 to ensure removal of non-specifically bound material.(5) Elution: Choose the elution method based on downstream application requirements.* A. Acidic Elution: Add 100 µL of Acidic Elution Buffer and resuspend the resin. Incubate at room temperature for 5 min. Centrifuge at 5,000 × g for 1 min. Carefully collect the supernatant without disturbing the resin. Neutralize immediately with Neutralization Buffer. 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. Store eluted samples at 4°C short-term or -20°C long-term.* C. Denaturing Elution (SDS-PAGE): Standard protein loading buffer (containing β-mercaptoethanol/DTT and SDS) will denature the anti-Flag antibody, releasing the bound protein but rendering the resin unusable for reuse. Add 20 µL of 2× Loading Buffer 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.Reagent CompatibilityReagentMaximum Tolerant ConcentrationNotesβ-Mercaptoethanol10 mMAvoid during purification; if used in IP, resin cannot be reusedDTT80 mMAvoid during purification; if used in IP, resin cannot be reusedSDS--Avoid during purification; if used in IP, resin cannot be reusedEDTA5 mMHigher concentrations reduce protein recoveryTween-205%High concentrations may reduce binding efficiencyTriton X-1005%High concentrations may reduce binding efficiencyNP-404%High concentrations may reduce binding efficiencyGuanidine HCl0.3 MHigher concentrations denature the antibodyUrea1.5 MHigher concentrations denature the antibodyGlycerol20%High concentrations may affect protein bindingNaCl1 MHelps reduce non-specific adsorptionTroubleshooting Guide... Read More | Amine-Reactive probe which passively diffuse into cells and it is nonfluorescent until the acetate groups are cleaved by intracellular esterases to yield the highly fluorescent, amine-reactive fluorophore. Upon reaction with amine-containing residues of intracellular proteins, these probes form dye Amine-Reactive probe which passively diffuse into cells and it is nonfluorescent until the acetate groups are cleaved by intracellular esterases to yield the highly fluorescent, amine-reactive fluorophore. Upon reaction with amine-containing residues of intracellular proteins, these probes form dye protein adducts that are well retained in cells as they move and divide during embryonic development.A Non-fluorescent cell permeant amine-reactive probe for long term tracing of cell... Read More | Product DescriptionEndo F2 cleaves N-linked (asparagine-linked) biantennary oligosaccharides from glycoproteins. It also will cleave high mannose glycans but at a 40x reduced rate. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, Product DescriptionEndo F2 cleaves N-linked (asparagine-linked) biantennary oligosaccharides from glycoproteins. It also will cleave high mannose glycans but at a 40x reduced rate. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact.Endoglycosidase F2 is less sensitive to protein conformation than PNGase F and is therefore more suitable for deglycosylation of native proteins. However, for optimal results, denaturation of the glycoprotein is recommended.Contents60 µl aliquot of enzyme (0.3 U) in 10 mM sodium acetate 25mM NaCl, pH 4.5Included with 20 µL and 60 µL pack sizes:5x Reaction Buffer – 250 mM sodium acetate, pH 4.5Molecular weight 32,000 daltonsSpecific Activity Defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micromole of denatured porcine fibrinogen in 1 minute at 37°C, pH 5.5. Cleavage is monitored by SDS-PAGE (cleaved fibrinogen migrates faster).Formulation The enzyme is provided as a sterile-filtered solution in 10 mM sodium acetate, 25mM NaCl, pH 4.5Specificity Endo F2 cleaves Asparagine-linked biantennary and high mannose glycans (at a 40X reduced rate). It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact. Endoglycosidase F2 is less sensitive to protein conformation than PNGase F and is therefore more suitable for deglycosylation of native proteins. However for optimal results, denaturation of the glycoprotein is recommended.Quality & Purity Endo F2 is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37°C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases.Stability Several days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly.Directions for use 1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 38 µl final volume with de-ionized water. 2. Add 10 µl 5x Reaction Buffer 4.5 3. Add 2.0 µl of Endo F2 to the reaction. Incubate 1 hour at 37°C. Monitor cleavage by SDS-PAGEThe production host strain has been extensively tested and does not produce any detectable glycosidases... Read More | Endothelin 3 (ET3) belongs to endothelin peptide family, which includes three members, ET-1, -2 and -3. These are 21-amino acid peptides, which are synthesized as precursors. They are converted to biologically active peptides, after being cleaved by proteases. There are two endothelin receptors Endothelin 3 (ET3) belongs to endothelin peptide family, which includes three members, ET-1, -2 and -3. These are 21-amino acid peptides, which are synthesized as precursors. They are converted to biologically active peptides, after being cleaved by proteases. There are two endothelin receptors called ETRA and ETRB, and ET3 binds to ETRB. It is localized to human intestine and colon.Application：Endothelin 3 has also been used as a ligand for endothelin receptor type B (EDNRB) in ex vivo enteric NCC (eNCC) migration assays. Endothelin 3 human, rat has been used for culturing neural tube explant culture, and the pharmacological study of endothelin receptors... Read More | Inorganic pyrophosphates are inevitably produced in the process of mRNA transcription in vitro. These substances have a great inhibitory effect on transcription. Inorganic pyrophosphatase (PPase) can hydrolyze the inorganic pyrophosphates produced in nucleic acid amplification experiments, promote Inorganic pyrophosphates are inevitably produced in the process of mRNA transcription in vitro. These substances have a great inhibitory effect on transcription. Inorganic pyrophosphatase (PPase) can hydrolyze the inorganic pyrophosphates produced in nucleic acid amplification experiments, promote the shift of reaction equilibrium to the product generation end, and increase the amount of products.The molecular weight of PPase (pyrophosphatase, inorganic, inorganic pyrophosphatase) is about 63kd, which can catalyze the hydrolysis of inorganic pyrophosphate to produce orthophosphate: P2O74_+H2O+PPase→2HPO42_. In the nucleic acid amplification experiment, PPase can hydrolyze the inorganic pyrophosphate generated with the reaction to avoid its inhibition on the reaction system. The removal of pyrophosphate can shift the reaction equilibrium to the product generation end.This product is a GMP level recombinant inorganic pyrophosphatase (yeast source) expressed by large-scale fermentation of E. coli. It is produced with raw and auxiliary materials of medicinal specifications, and the host protein residue and nucleic acid residue are strictly controlled. The product production and quality management procedures in line with GMP specifications ensure that the production process and all raw and auxiliary materials can be traced.Quality requirements project standard appearance Clear liquid Visible foreign matter Compliance with regulations PH value 7.5±8.5 activity 98U/ml-102U/ml purity ≥95% Endonuclease residues Degradation of substrate shall not exceed 10% Exonuclease residues Degradation of substrate shall not exceed 10% RNase residue Degradation of substrate shall not exceed 10% Bacterial endotoxin content ≤10EU/ml Exogenous DNA residue ≤100pg/mg Host protein residue ≤50ppm Mycoplasma detection negative Heavy metal residues ≤10ppm Follow the following specifications1. ISO 9001:2015, certified facility。2. GMP appendix - cell therapy products State Drug Administration.3. general introduction to human gene therapy - Chinese Pharmacopoeia 2020, National Pharmacopoeia Committee.4. USP chapter <1043>, adjuvant materials for cell, gene, and tissue engineered products.5. USP chapter <92>, growth factors and cytokines used in cell therapy manufacturing.6. Ph. Eur. General chapter 5.2.12, raw materials of biological origin for the production of cell-based and gene therapy medical products.Product features1. hydrolyze inorganic pyrophosphate.2. DNA synthesis: significantly enhance DNA replication ability.3. RNA synthesis: increase RNA production in in vitro transcription reaction.4. The optimal reaction temperature is 25℃, and the enzyme can be inactivated at 65℃ for 10min.Product usage1. optimize RNA transcription: improve the RNA yield of in vitro transcription reaction.2. remove PPI contamination from reagents for SNP genotyping by pyrophosphate assay.3. promote the synthesis of protein, RNA and DNA.4. catalyze the reaction of PPI + H2O → 2pi.5. ssr-pcr optimization：Improve efficiency and increase DNA production.Activity definitionCatalytic inorganic pyrophosphate formation 1 per minute under standard reaction conditions µ The amount of enzyme required for mol phosphate was defined as 1 active unit.Preservation system20 mM Tris-HCl; 100 mM NaCl; 1 mM DTT; 0.1 mM EDTA; 50% (v/v) Glycerol; pH 8.0。 Storage temperature-20±5 ℃。Matters needing attention1. the enzyme has activity in various reaction buffers. Generally, the enzyme can be directly added in HDA, lamp and other experiments.2. the dosage of the enzyme needs to be optimized in different experiments, usually adjusted at the concentration of 0.05~1u/ml.3. the optimum reaction temperature of the enzyme was 25 ℃, and it was active at 16~37 ℃, and the enzyme could be inactivated at 65 ℃ for 10min.4. cofactor: mg2+ is necessary for enzyme activity... Read More |