Analysis of Recombinant Adeno-Associated Virus (rAAV) Purity Using Silver-Stained SDS-PAGE

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Adeno-associated virus (AAV) virions comprise VP1, VP2, and VP3 capsid proteins.
• These proteins are typically present in a 1:1:18 ratio within the virion.

Purpose of the Study:

• To describe a protocol for determining the purity of recombinant adeno-associated virus (rAAV) preparations.
• To outline a method for estimating the particle concentration of rAAV vectors.

Main Methods:

• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation.
• Silver staining for visualizing protein bands on the gel.
• Utilizing a known particle titer of a purified rAAV preparation.

Main Results:

• VP1, VP2, and VP3 proteins should be the only visible bands in purified rAAV preparations.
• Expected migration of VP1, VP2, and VP3 at approximately 87, 73, and 62 kDa, respectively.
• SDS-PAGE and silver staining confirm rAAV purity and enable semi-quantitative titer estimation.

Conclusions:

• SDS-PAGE and silver staining are effective for assessing rAAV purity.
• This technique provides a reliable method for estimating rAAV particle concentration.