Hi,

As you mentioned the pH of sample appears problematic for TCA precipitation then centrifugal filters would be the best option for you to concentrate your protein. But before that you have to dialyzed your lysate using specific kDa filter according to molecular weight of your protein to reduce the contaminants level in your sample.

The Protein Man Says:

Protein Concentration is an important step in protein extraction and purification. The large amount of starting material used to acquire an adequate quantity of a protein of interest results in large volumes of dilute protein. Following protein extraction, the purified proteins need to be concentrated.

Several methods exist for protein concentration.

https://info.gbiosciences.com/blog/bid/120657/protein-concentration-alternative-methods-to-ultrafiltration?hs_amp=true

Dialysis. One of the popular approaches is to use a dialysis technique. The dilute protein solution is placed in a dialysis bag or alternative dialysis device, such as Tube-O-DIALYZER, and the sample is dialyzed against a water absorbing polymer. The dialysis membrane retains the proteins inside the dialysis bag or device and the sample is concentrated as the water is pulled across the dialysis membrane. An ideal water absorbing polymer would be polyethylene glycol. G-Biosciences offers both a Concentrator Solution and a Concentrator Powder for this purpose. Our Tube-O-Concentrator contains the liquid concentrator and dialysis devices for added convenience https://info.gbiosciences.com/blog/bid/120657/protein-concentration-alternative-methods-to-ultrafiltration?hs_amp=true

If you just need to run your samples on gel, I would suggest to simply precipitate them with TCA. When you add loading buffer with bromophenol it will turn yellow indicating the low pH. Then you can easily bring your pH up by adding tris or NaOH. The sample will turn blue again.This will be even suitable for comparative WB as long as you load equal amounts of initial lysate. In summary, my pipeline here would be: 1) clarify the lysate by centrifugation; 2) adjust total protein amounts using Bradford; 3) TCA precipitation of aliquotes that contain equal amounts of protein in the equal volumes (protein concentration as well as the sample volume affect TCA precipitation efficiency); 4) add loading buffer, adjust the pH, and load everything on gel.

Hope this helps.

In protein isolation, proteomic, or peptidomic procedures, protein solutions are often concentrated to remove solvents and undesirable molecules, to separate protein fractions or to increase protein concentrations. Proteins can be concentrated by precipitation from solution with ammonium sulfate, polyethylene glycol, organic solvent, trichloroacetic acid, potassium chloride/sodium dodecyl sulfate, and three-phase partitioning. Solvents can be removed by passage through a semipermeable barrier where protein solutions are forced against the barrier in a centrifuge tube or with increased pressure concentrating protein in the remaining solution. The semipermeable barrier can be surrounded by a hygroscopic reagent to draw the solvent across the membrane. Proteins can be concentrated by freeze-drying (lyophilization). All these methods to concentrate proteins are discussed. https://link.springer.com/protocol/10.1007/978-1-4939-2718-0_2

If you need your sample for electrophoresis only, then chloroform/methanol precipitation (doi:10.1016/0003-2697(84)90782-6) is your best bet. If you decide to use TCA instead, you should extract the pellet with ether to remove TCA, only neutralising it will increase the salt concentration of the sample, which may interfere with proper stacking. For safety, replace the diethylether by tert-butylmethyl ether (MTBE), to avoid the formation of explosive peroxydes. The other hazards (fire, endocrine disruptor) still need to be managed, though.

if you want to treat your proteins nicely and your volume is not too large for convenient dialysis, I would suggest dialysis into the buffer of your choice followed by dehydration using a concentrating resin. Aquacide from CalBiochem or Sigma-Adrich and Bio-Rads Bio-Gel concentrating resin are very good examples. Just bury your membrane in the solid and it absorbs the water.

It can get messy bit its reasonable quick and you will still have native structured proteins to work with.

Can you add information on the volume of the samples you want to concentrate for electrophoresis. Are we talking about 10, 100 or 1000 ml that you need to concentrate to produce a sample for electrophoresis.

You mentioned "secreted proteins". These may be very dilute. A good approach is to bind them to an ion exchange resin. These usually have a high capacity for protein binding and allow elution in a smaller volume than has been loaded.