Hi,

if your protein absorbs UV (the peak on FPLC you mentioned) than it should have some tryptophan residues. This allows you to use stain-free gels (BioRad for example) or you can prepare such yourself using trichloroethanol. You can also use silver staining, but its laborious and tricky.

Good luck!

Thanks Anton Slyvka that's really helpful - I didn't realise that's how the stain-free gels worked. I'm going to order some now!

We tried silver staining and though it worked as you say it is really laborious and we struggled with the reproducibility

Dear Joseph Philip Bennett ,

In addition to what is said by Anton Slyvka I would like to add some suggestions of what might be going on here. Your observation seems somewhat strange since it has been suggested that a highly Ala rich protein could be stained easily according to the following paper:

Farmer, R. S., & Kiick, K. L. (2005). Conformational behavior of chemically reactive alanine-rich repetitive protein polymers. Biomacromolecules, 6(3), 1531-1539.

However the following paper indeed indicated some problems that might be similar like yours:

Breibeck, J., & Skerra, A. (2018). The polypeptide biophysics of proline/alanine‐rich sequences (PAS): Recombinant biopolymers with PEG‐like properties. Biopolymers, 109(1), e23069.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/bip.23069

They state that you can observe migration to higher MW presumably because of poor binding of SDS due to low amount of hydrophobic amino acid side chains. They also refer to lack of binding to CBB to similar type of proteins you talk about, as described in:

Article PASylation: A biological alternative to PEGylation for exten...

They (Breibeck & Skerra) refer to a BaI2 staining that might be worthwhile checking.

It might also be worthwhile to check some improved CBB staining methods:

Georgiou, C. D., Grintzalis, K., Zervoudakis, G., & Papapostolou, I. (2008). Mechanism of Coomassie brilliant blue G-250 binding to proteins: a hydrophobic assay for nanogram quantities of proteins. Analytical and bioanalytical chemistry, 391(1), 391-403.

Pink, M., Verma, N., Rettenmeier, A. W., & Schmitz‐Spanke, S. (2010). CBB staining protocol with higher sensitivity and mass spectrometric compatibility. Electrophoresis, 31(4), 593-598.

See also for more great practical tips:

Dyballa, N., & Metzger, S. Fast and Sensitive Coomassie Staining in Quantitative Proteomics.

Hope this helps you a bit further.

Best regards.