Editing Expression of HA NA using Baculovoirus (section)

== Characterization and Quality Control ==

1. Measuring protein concentration<br> Take an aliquot of the concentrate and measure it by using your protein quantification method of choice. Set protein concentration to 1 mg/ml by adding PBS and freeze small aliquots to -80 °C. Freeze thaw cycles harm the protein structure and can lead to aggregation.

<br> 2. SDS-PAGE and Coomassie staining<br> Run 500 ng of your protein on a SDS-PAGE and perform a Coomassie staining. For fast and convenient staining use Coomassie reagent in combination with a microwave protocol. HA proteins typically appear at 64 kDa whereas NA proteins should run at a size of around 56 kDa. Using the described protocol you should not see any degradation products. If degradation occurs it is usually a sign of yeast/fungal contamination during expression. In this case repeat the procedure and make sure to keep everything sterile.

<br> 3. Western blot/ELISA<br> In order to verify protein identity we recommend do to a Western blot or ELISA assay with a specific antibody. Ideally, ELISA assays are performed with an antibody that binds to conformational epitopes (e.g. anti-HA stalk antibodies1). Using such an antibody the correct folding of the protein can be confirmed.

<br> 4. Measuring NA activity<br> Correct folding of the viral NA can also be determined by measuring NA activity. We recommend to perform this test using the commercially available NA*STAR assay.

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[[Category:Protocols]]