Hi! Recently I attempted a two step protein purification (affinity- GSTrap FF resin then iex- HiTrap Q FF) for my recombinant protein.

I connected two 1ml prepacked resin and injected 3mg of crude protein. Wash with 10CV buffer then elute in step with 5CV each. My target protein with tag is around 75kDa and usually when I used 1ml single prepacked resin (injected crude 1.5mg) I get a thick band at the 75kDa mark with fine band right below it as shown in Figure 1. This time around I got two set of band at the 50 kDa mark and 25kDa mark implying my target protein (thicker bands at 55kDa) and tag (thinner bands at 26kDa) (Figure 2). I used to think that this is caused by an old Triton X-100 that I incorporated in the buffer but even when I exclude it the issue persist.

2. IEX -HiTrap Q FF

Ive tried to separate the band via IEX. However, the now thicker band (previously at the 75kDa mark in affinity PAGE gel) has now become thinner and vice versa (Figure 3). For the record, when I use 1ml affinity resin for capturing step instead of 2ml I yield a single band at 75kDa.

Apologies for the very faint band my phone couldnt capture it nicely but Ive labeled them hopefully you can see it better.

My question is does the connected resin has damaged my protein in any way that cause them to sort of cleave? or is this caused by other reason? Any advice is very much appreciated.

Thank you in advance!