I am conjugating Amyloid-beta (Abeta) peptide (MW ~4500) with a small molecule (starting with monomeric abeta). I tried purifying the product with protein concentrator with 3000 molecular weight cut-off (MWCO), where after centrifuge, unreacted small molecule should come down (filtrate) with a solvent. But, purification is not good as small molecule has mass around ~800. I have very low quantity in microgram scale so didn't try FPLC. In the literature people have shown P-10 column use but not reported any exact protocol. If anyone has detailed protocol for the purification of such peptide, please share with me. Thank you.
The best methods I have seen thusfar are the use of capillary electrophoresis and thermophoresis.
https://www.nature.com/articles/s41592-019-0451-6
Article Field amplified sample stacking of amyloid beta (1-42) oligo...
For a small amount of a small peptide, you could use UPLC with either reverse phase or size exclusion chromatography (SEC). If you want to avoid exposure to organic solvents, SEC is preferable.
Also simple method is dialysis (cut off ≥ 2 kDa)
https://www.thermofisher.com/ru/ru/home/life-science/protein-biology/protein-purification-isolation/protein-dialysis-desalting-concentration/dialysis-products/slide-a-lyzer-mini-dialysis-devices.html
We routinely use MWCO filter of 3000 Da for our proteomics work and never had issue. Did you make sure that the the mass of 800 you are seeing is not a peptide/fragment of the beta amyloid conjugate? What is the charge on that molecule? Sorry if i did not get your question right. How long and at what speed did you centrifuge to get the filtrate? Did you wash them a couple of times before you put your sample in?
You want to separate a 4.5 kDa Peptide and a 5.3 kDa conjugate from an 800 Da unreacted label, or even the unconjugated peptide from the conjugate? If size is your separation criterium, you'll need a reasonably long column filled with high-resolution matrix with appropriate selectivity and low flow rates, see https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/General_Information/1/ge-gel-filtration.pdf for protocols.
PD-10 columns (http://wwwuser.gwdg.de/~jgrossh/protocols/protein-purification/PD10.pdf) are disposable plastic columns filled with 10 mL Sephadex G25. They are used to elute proteins larger than the size limit (25 kDa) with the void volume, whilest small molecules (like 125I-) stay on the column and can be disposed with it. They are unlikely to work in your case, even Spintrap G-10 columns (filled with Sephadex G10) are probably not selective enough.
Other separation principles (ion exchange, reverse phase) can be used for chromatography and may be more promising, I have discussed chromatography in detail in doi:10.1007/978-1-4419-7251-4_14. For very small sample sizes, HPLC may be more suitable than FPLC due to smaller column size.
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