We are working with a protein with a MW of 12 kDa with a C-terminal "aldehyde-tag" (formyl-glycine) produced as described in:
https://pubmed.ncbi.nlm.nih.gov/17450134/
After purification and initial concentration on VivaSpin 20 (PES) MWCO 5000 spin filters, we attempted to fluorescently tag the protein using an aminooxy-coupled fluorescent dye in 0.1 M MES pH 5.5. We conjugated in 100 µL with 10 µM protein (equivalent to ~12 µg protein). The protein was diluted from its stock in 0.1 PBS pH 7.4 in milliQ water, and then spiked with 1M MES pH 5.5 (10 µL 1M MES in 90 µL diluted protein). However, when we attempted to purify the protein after tagging on VivaSpin 500 (PES) MWCO 5000 spin filters, all the protein was gone (verified on both NanoDrop 2000 and SDS-PAGE). We saw no protein in the flow-through, either.
In a second attempt, we used 100 µL the protein in the concentration of the stock (~48 µM ≈ 56 µg), and then included a buffer exchange into 0.1 M MES pH 5.5 using VivaSpin 500 (PES) MWCO 5000 spin filters. To assure ourselves that the protein concentration was still comparable to the original, we checked on both NanoDrop 2000 and SDS-PAGE. On the NanoDrop, we could not detect the protein and on the SDS-PAGE the intensity of the band was visibly lower than that of the original stock solution. We suspected that the filter may be faulty, and this repeated the buffer exchange in VivaSpin 6 (PES) MWCO 5000 spin filters with similar results. Still no protein in the flow-through, either. These specific spin filters have been used for the same protein without the aldehyde-tag in our lab with little to no protein loss.
According to Sartorius, they achieve at least 30-50% yields starting with 250-500 ng in untreated spin filters:
https://www.vivaproducts.com/downloads/vivaspin-app-notes-3.pdf
We did try membrane passivation with 5% TWEEN-20 (v/v), however this just completely clogged our spin filter, hampering flow-through even at dilute solutions of our protein.
We have considered 2 scenarios:
1. Since the 0.1 M MES pH 5.5 is the reaction buffer for the dye conjugation, is it possible that the aldehyde-tag is capable of reacting with the PES membrane under these conditions?
2. The protein we are working with is pretty compact and globular in nature, so maybe the protein is capable of squeezing into the pores of the PES membrane, but incapable of passing through?
We see close to no flow-through when we reach lower volumes / higher concentrations - even with centrifugation at 15 minute intervals with aspiration of the remaining solution above. However, could this not could be caused by both scenarios, since they both would clog the membrane?
How much protein are the PES membrane capable of absorbing?
My organic chemistry is not good enough to verify scenario 1, but I have not found any direct evidence of it. Can someone confirm or deny whether the aldehyde to PES reaction is possible in aqueous solution at pH 5.5?
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