I am trying to concentrate a 43kDa soluble protein expressed in mammalian cells. I tried to concentrate it but the sample went through the column very quickly even at centrifugation speed lower than recommended. I wonder if there was aggregation or binding of the protein to the filter. Any suggestion to prevent non-specific binding and how to avoid losses?
Thanks
Hi there,
What is the cut off of the membrane? Did you check the content of the retentate and filtrate? According to what you wrote I would say either the membrane has cracked or the cut off is far larger than 43kDa (in both cases absorbance in retentate and filtrate should be very similar...)
Hi Dominique,
Thanks for the answer, it was 10 MWCO, yes I did check it, the thing is it was my very first time and I centrifuged the protein sample for 5 minutes slower than the recommended speed. out of 2ml, I recovered around 75 ul, I had no idea about aggregation or non-specific binding to the filter and before I get to know I already had washed the column. I have checked the concentration of the recovered protein but there was not a big change compared to the initial concentration and nothing was detected on the gel. That,s why I am looking for suggestions
Thanks once again
So there was no concentration of the protein during the process. Did you check the filtrate? If nothing in the filtrate your protein tends to stick to the plastic/membrane. If protein in the filtrate then it's membrane cracking problem.
Thanks again Dominique,
I guess protein got stuck to the filter and I am looking for recommendations how to avoid that?
So, your protein is not detected in the filtrate or washes, nor is it present in the retentate. Thus, you conclude it's stuck to the filter, correct? Is the material being applied to the column a relatively pure solution of your protein, or is it a more complex mixture? What type of buffer is it in (salt/no salt, detergents, pH, etc.)? How much total protein is in your sample?
Do you know what type of membrane was in your Vivaspin? If it's a Vivaspin 2 (2 ml capacity), it's offered with 3 different membrane configurations: Polyethersulfone (PES), Cellulose triacetate (CTA) and Hydrosart.
Regardless of the membrane chemistry, I find it hard to believe that your protein was completely (100%) lost due to non-specific binding, unless you had very small amounts of the protein to begin with. If you do have small amounts of the purified protein and you still wish to use the spin concentrator (rather than SpeedVac or lyophization), you should pretreat the membrane - it's called surface passivation. One protocol involves pretreating the entire filtration unit overnight with 5% Tween-20 (v/v) in MQ/distilled water (ultrapure H2O), then soaking/rinsing with a couple of changes of MQ water. The unit can then be used immediately or stored at 4oC with MQ water keeping the membrane wet.
What was the initial concentration? It could be that you passed the solubility limit of your protein by concentrating 25 times an your protein simply gelled up on the filter. The disadvantage with speed-vac concentration is that you will also be concentrating salts.
Do check the expiry dates of concentrators. Ir expired, there is a fair chance that the membrane does not maintain its original properties and allows everything(including your protein) to pass through.
Thanks for your answer Jhon,
I haven't lost all of the protein but the recovery rate was really low, fractions were getting detected by SDS-PAGE before and not the retentate later on. There was a minor change in concentration of the retentate. I am working with viral glycoproteins and purified fraction contain tris and glycine-HCl. Vivaspin column used was of 2ml capacity was used. Thanks for the recommended protocol, I will go for it
Your protein might precipitated in the Vivaspin column. After concentration spin , remove the retentate ,then try 4~6M Urea contained in your Tris, glycine-HCL solution to rinse the Vivaspin column, to see if you could recover your target protein.
Is your protein a protease . Or is there a protease contaminant inn your sample.
As suggested by John Mcgrath, passivation of the membrane can reduce adsorption of your protein. For passivation I use 2% BSA (maximum volume, centrifugation at the recommended speed) and I rince several times with my buffer before loading the sample.
Hi Emeline,
How can I minimize the chances of contamination after using BSA, because I used 1%BSA and washed the column a couple of times, I was happy to see protein has been concentrated after Nano-drop but found a 66kDa band of BSA on my gel either.
In case I am trying to turfy a protein of ma molecular weight lower than BSA how to minimize chances of BSA contamination
Thanks
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Proteins