Zeba buffer exchange low protein yield - (Feb/10/2016 )

Hi everyone. I use 2, 5, and 10 mL Zeba spin desalting columns from Thermo to buffer exchange proteins as part of my work. Sometimes the yield is great (>95%), but other times it's really bad (<75%). Does anyone know what could be at fault or investigations I could run to debug?

Thank you!

Michael

I use the same columns.  Are you exchanging consistent volumes each time and are those volumes near the low or high end of the range?

Missle on Wed Feb 10 18:07:37 2016 said:

I use the same columns.  Are you exchanging consistent volumes each time and are those volumes near the low or high end of the range?

The volume varies, but it is always within the recommended range in the manufacturer protocol. And I use the stacker when necessary, too. The volume doesn't change that much over the buffer exchange, so I am thinking that protein gets stuck in the column when it shouldn't. It's size exclusion, right? With a 7K cutoff.

I check the protein concentration via BCA in duplicates with duplicate standards before and after the buffer exchange.

So the yield is consistently good for you?

The yield consistency is good within the same protein.  If the protein contains aggregates, those aggregates can get basically filtered out by the resin.  This has happened to me numerous times when buffer exchanging post conjugations or with proteins that have solubility problems.  For non-aggregated proteins, the yields have been fine but I do notice lower yields when working on the lower end of the volume range, even with a stacker - but it's never been lower than ~80-85% recovery due to low-end volume.

Aggregates would be my first guess.  Back when Pierce was Pierce, they had good tech support so maybe that has carried over now that it is a Thermo product and you could try them? 

Missle on Wed Feb 10 18:47:43 2016 said:

The yield consistency is good within the same protein.  If the protein contains aggregates, those aggregates can get basically filtered out by the resin.  This has happened to me numerous times when buffer exchanging post conjugations or with proteins that have solubility problems.  For non-aggregated proteins, the yields have been fine but I do notice lower yields when working on the lower end of the volume range, even with a stacker - but it's never been lower than ~80-85% recovery due to low-end volume.

 

Aggregates would be my first guess.  Back when Pierce was Pierce, they had good tech support so maybe that has carried over now that it is a Thermo product and you could try them? 

In size exclusion chromatography, though, shouldn't large molecular weight stuff (like aggregates) flow through? Or, you think if it's really large, it is not even soluble and gets stuck at the top of the column?

Sorry it's taken me so long to reply.  Large enough aggregates (approaching macroscopic) will wither be caught by the resin or trapped on the frit.

I should be able to see the aggregates as precipitate in the sample tube before running SEC, though, yeah? I'll let it sit a while next time and check before running the Zeba.