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In-vitro Mutagenesis01:16

In-vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.

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Reliable Detection of SGLT2 Protein by Knockout-Based Antibody Characterization.

Takuo Hirose1,2, Hiroki Ito1, Akari Endo1

  • 1Division of Nephrology and Hypertension (T.H., H.I., A.E., T.K., K.Y., R.I., A.K., I.O.-Y., W.Y., T.M.), Faculty of Medicine, Tohoku Medical and Pharmaceutical University, Sendai, Japan.

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Summary

Reliable detection of sodium-glucose cotransporter 2 (SGLT2) protein is crucial for research. The antibody HPA041603 demonstrated consistent SGLT2 detection in rodent and human kidneys, unlike other tested antibodies.

Keywords:
animalsglucosehumanskidneysodium

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Area of Science:

  • Nephrology
  • Biochemistry
  • Immunohistochemistry

Background:

  • Sodium-glucose cotransporter 2 (SGLT2) plays a key role in renal glucose reabsorption.
  • Pharmacological inhibition of SGLT2 offers cardio- and renoprotective effects.
  • Accurate detection of SGLT2 protein is hindered by antibody specificity concerns.

Purpose of the Study:

  • To evaluate the specificity and reliability of commercially available anti-SGLT2 antibodies.
  • To identify suitable antibodies for detecting SGLT2 protein in experimental and translational studies.

Main Methods:

  • Immunohistochemistry and Western blotting were employed to assess antibody performance.
  • Kidney and heart tissues from wild-type and SGLT2-deficient mice and rats were used.
  • Human kidney tissues, including renal cell carcinoma samples, were also examined.

Main Results:

  • Antibodies ab306558 and HPA041603 showed specific immunostaining in rodent kidneys.
  • HPA041603 demonstrated specific SGLT2 detection in human kidney proximal tubules, absent in tumor areas.
  • Western blotting confirmed SGLT2-specific bands at ~55 kDa with certain antibodies, shifting to ~45 kDa after deglycosylation.

Conclusions:

  • The antibody HPA041603 consistently and specifically detected SGLT2 in both rodent and human kidney tissues.
  • Other evaluated antibodies exhibited limited specificity for SGLT2 detection.
  • Antibody validation using knockout models and optimized conditions are vital for accurate SGLT2 expression analysis.