3X HA Tag |
| Catalog No.GC72280 |
3X HA Tag es un péptido biológico activo.
Products are for research use only. Not for human use. We do not sell to patients.
Sample solution is provided at 25 µL, 10mM.
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3X HA Tag is a bioactive peptide composed of the 9-amino-acid sequence YPYDVPDYA repeated 3 times[1]. HA Tag is derived from the hemagglutinin (HA) surface glycoprotein of human influenza virus, featuring high specificity and hydrophilicity. It enhances the possibility of antigen - antibody reactions on the surface of fusion proteins[2]. With more repeats, 3X HA Tag has stronger antigenicity and higher binding affinity for anti - HA antibodies, ensuring higher detection sensitivity, especially for low - abundance proteins[3]. It's widely used in protein affinity purification experients to elute HA-tagged proteins[4][5].
References:
[1] Schneider, B. L., Seufert, W., Steiner, B., Yang, Q. H., & Futcher, A. B. (1995). Use of polymerase chain reaction epitope tagging for protein tagging in Saccharomyces cerevisiae. Yeast (Chichester, England), 11(13), 1265–1274.
[2] Zhao, X., Li, G., & Liang, S. (2013). Several affinity tags commonly used in chromatographic purification. Journal of analytical methods in chemistry, 2013, 581093.
[3] Ensinck, M., De Keersmaecker, L., Heylen, L., Ramalho, A. S., Gijsbers, R., Farré, R., De Boeck, K., Christ, F., Debyser, Z., & Carlon, M. S. (2020). Phenotyping of Rare CFTR Mutations Reveals Distinct Trafficking and Functional Defects. Cells, 9(3), 754.
[4] Lim, J., Iftner, T., & Simon, C. (2021). Native Isolation of 3×HA-Tagged Protein Complexes to Characterize Protein-Protein Interactions. Current protocols, 1(2), e29.
[5] Tanaka, T., Umemori, T., Endo, S., Muramatsu, S., Kanemaki, M., Kamimura, Y., Obuse, C., & Araki, H. (2011). Sld7, an Sld3-associated protein required for efficient chromosomal DNA replication in budding yeast. The EMBO journal, 30(10), 2019–2030.
Protocol for 3xHA tag for protein purification[1]：
A: Cell Harvesting and Lysis
1. Harvest YTT4 cells (7×10⁷ cells/ml, 2.5L) expressing Sld7–3Flag–HA.
2. Wash cells once with distilled water.
3. Disrupt cells using a mortar grinder under liquid nitrogen.
4. Suspend the cell lysate in 5ml of lysis buffer A (50mM HEPES–KOH (pH 7.5), 300mM KCl, 0.5% Tween 20, 0.05% NP40, 10% glycerol, 1×Complete, 1% protease inhibitor cocktail, 50mM NaF, 2mM b-glycerophosphate, 0.4 mM Na3VO4, 0.5 mM Na4P2O7, and 1mM PMSF).
5. Clarify the lysate by centrifugation at 12,000rpm at 4°C for 15 minutes.
B: Protein Extraction and Adsorption
6. Adsorb the protein extracts to 1ml of Sepharose 4B at 4°C for 30 minutes.
7. Incubate the mixture with 1ml of anti-Flag M2 affinity gel, preincubated with lysis buffer A containing 5mg/ml BSA, at 4°C for 2.5 hours.
C: Washing and Elution with Flag Peptide
8. Wash the beads three times with ml of lysis buffer A containing 5mg/ml BSA.
9. Elute the bound proteins twice by incubating the beads with 500μl of Flag elution buffer (lysis buffer A containing 150mg/ml of 3× Flag peptide) at 4°C for 1 hour.
D: Further Purification with Anti-HA Matrix
10. Mix the eluted solution with 120μl of anti-HA matrix, pretreated with anti-Flag M2 affinity gel, and incubate at 4°C for 2.5 hours.
11. Wash the beads three times with 1 ml of buffer A containing 5mg/ml BSA, once with 1 ml of buffer A containing 0.1mg/ml BSA, and once with 1ml of buffer A.
12. Elute the bound proteins by incubating the beads twice with 100μl of HA elution buffer (lysis buffer A containing 1.5mg/ml 3× HA peptide) at 37°C for 30 minutes.
E: Protein Precipitation and Analysis
13. Precipitate the proteins by adding trichloroacetic acid and subsequent centrifugation.
14. Boil the precipitate for 3 minutes in 50μl of 1× SDS loading buffer.
15. Separate the proteins in an SDS–polyacrylamide gel and stain with SilverQuest.
16. Excise the protein band from the gel, digest with trypsin, and analyze by mass spectrometry.
This protocol only provides a guideline, and should be modified according to your specific needs.
References:
[1] Tanaka, T., Umemori, T., Endo, S., Muramatsu, S., Kanemaki, M., Kamimura, Y., Obuse, C., & Araki, H. (2011). Sld7, an Sld3-associated protein required for efficient chromosomal DNA replication in budding yeast. The EMBO journal, 30(10), 2019–2030.
| Cas No. | SDF | ||
| Formula | C205H272N38O67S | M.Wt | 4372.63 |
| Solubility | DMSO : 100 mg/mL (22.87 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO) | Storage | Store at 2-8°C,Sealed storage, away from moisture and light |
| General tips | Please select the appropriate solvent to prepare the stock solution according to the
solubility of the product in different solvents; once the solution is prepared, please store it in
separate packages to avoid product failure caused by repeated freezing and thawing.Storage method
and period of the stock solution: When stored at -80°C, please use it within 6 months; when stored
at -20°C, please use it within 1 month. To increase solubility, heat the tube to 37°C and then oscillate in an ultrasonic bath for some time. |
||
| Shipping Condition | Evaluation sample solution: shipped with blue ice. All other sizes available: with RT, or with Blue Ice upon request. | ||
| Prepare stock solution | |||
|
1 mg | 5 mg | 10 mg |
| 1 mM | 0.2287 mL | 1.1435 mL | 2.287 mL |
| 5 mM | 0.0457 mL | 0.2287 mL | 0.4574 mL |
| 10 mM | 0.0229 mL | 0.1143 mL | 0.2287 mL |
Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)
g
μL
Step 2: Enter the in vivo formulation (This is only the calculator, not formulation. Please contact us first if there is no in vivo formulation at the solubility Section.)
Calculation results:
Working concentration: mg/ml;
Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )
Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O, mix and clarify.
Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.
Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such as vortex, ultrasound or hot water bath can be used to aid dissolving.
3. All of the above co-solvents are available for purchase on the GlpBio website.
Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
Average Rating: 5 (Based on Reviews and 30 reference(s) in Google Scholar.)
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