8/5/21

Dear Carolyn,

What you are describing is apparently a modification of the technique known as Western Blot. Most of the time, I believe, it is done to detect soluble proteins that have been resolved by polyacrylamide gel electrophoresis (PAGE). After separation by PAGE, the proteins are transferred to nitrocellulose (NC) via electroelution. A primary antibody (Ab) specific for the protein of interest is applied to the NC and allowed to bind to the protein. Excess Ab is washed off the NC and the NC is washed w/ an inert protein, e.g., BSA or casein (skim milk is commonly used) to block sites on the NC that bind proteins non-specifically. A secondary Ab that recognizes the primary Ab is then applied and specific binding occurs. The NC is washed again to remove unbound sec. Ab. The sec Ab is covalently bound to an enzyme, e.g., peroxidase or alk. phosphatase. A chromogenic (or fluorometric) substrate for the enzyme is added. When broken down by the enzyme, a colored product is released form the substrate in the region of the NC where the protein of interest is localized. The color (or fluorescence) can be measured to provide quantitation of the protein.

I have not heard of immobilizing intact cells on NC, but I suppose it can be done. If so, the procedure should work for you. If your Pichia cells secrete the protein of interest in soluble form, you can remove the cells (e.g., centrifugation), recover the protein and do the detection as described above. Without the cells, the detection process would probably be "cleaner".

There are lots of sites on the internet to guide you in performing a Western Blot.

I hope this information helps you.

Bill Colonna Ames, IA [email protected]

Check https://www.mdpi.com/2079-6382/9/9/541/pdf

Kindly look at this also

https://www.mdpi.com/1422-0067/22/7/3677/pdf