Hi,

If your laboratory is capable to perform, I strongly suggest freeze-drying. It keeps the protein sample stable and makes it concentrated. Protein precipitation using different kosmotropes (e.g. TCA, sulfate) is also a good alternative way to reconcentrate.

PES or CA membranes are not prone to be retentive typically for protein applications. Have you tried to use different spin columns (brand, MWCO, or material)?

If your peptide or protein is not a very sticky one, then you may consider the investigate protein concentration at your filtrate instead of retentate after centrifugal membrane application. If you share the MWCO of your CA membrane, it would be easier to decide whether it is a co-elution or adsorption of your target.

There are also specific or non-specific magnetic bead (nanoparticles) isolation/extraction and enrichment procedures for this purpose, you may look for a proper one depending on your protein major affinity.

If it is a real adsorption problem, non-specific binding (NSB) applications like adding analyte protectants (e.g.BSA) or adding solubilizing agents (e.g chaotropes) may help.

You may also have a look at the link below for further discussion;

https://www.researchgate.net/post/Can_anyone_suggests_a_good_method_to_reconcentrate_proteins_after_isolation_Precipitation_Speedvac_or_Lyophilization

Good luck,

Emir...

Dear Soi Yun,

I suggest you suggest you to see links and attached files on topic.

http://www.protocol-online.org/biology-forums-2/posts/27135.html

https://www.sartorius.com/download/501692/5/vivaspin-concentration-peptides-applicationnote-210x280mm-data.pdf

https://royalsocietypublishing.org/doi/10.1098/rsos.181433

https://www.sciencedirect.com/science/article/abs/pii/S1872204016609490

https://www.hindawi.com/journals/bmri/2011/245291/

Best regards

uI suggest you follow this discussion : https://www.researchgate.net/post/Which_are_the_best_methods_to_quantify_peptide_concentration_for_mass_spectrometry

Maybe you could try freeze-drying the protein.

İsmail Emir Akyildiz Thank you for your reply :D.

I used Amicon 3K centrifuge filter unit (consisted with CA membrane). Because my sample's MW is around 17 kDa.

Actually, we found another problem with buffer.

We tested various buffer to solve the non-specific adsorption problem that I asked.

The buffer we tested...

50 mM sodium phosphate, 150 mM NaCl, pH 8 and 7.4

50 mM Tris-HCl, 150 mM NaCl, pH 7.4

50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA pH 7.4

50 mM Sodium acetate, pH 5.5

But my sample has aggregates in all of the buffer conditions during the dialysis for buffer exchange.

But when we dialyzed with 50 mM Sodium acetate, pH 5.5, the small white particles were observed. When we sonicated it for 20-30 min at RT, the particles were removed and not observed again.

So we think the loss of our sample is occurred by the peptide aggregation. It is tried to aggregate when is concentrated with a centrifuge filter. And when we filter the sample, the aggregates were removed with filter pore.

I can try your approach after solve the aggregation problem.

Thank you so much :D

Mohamed-Mourad Lafifi Thank you for your attachments :D.

You are welcome and many thanks for sharing how your work proceeds,

I hope the followings links/pieces of information and discussions would help to you based on aggregation inhibition request;

https://info.gbiosciences.com/blog/tips-for-preventing-protein-aggregation-loss-of-protein-solubility

https://www.researchgate.net/post/How_can_I_prevent_protein_aggregation

https://www.researchgate.net/post/Is_there_any_suggestion_to_avoid_protein_aggregation_during_purification

https://www.researchgate.net/post/Is_there_any_suggestion_to_avoid_protein_aggregation_during_purification

https://www.biozentrum.unibas.ch/fileadmin/redaktion/05_Facilities/01_Technology_Platforms/BF/Protocols/Preventing_Protein_Aggregation.pdf