I am looking to blot for secreted proteins from cells in suspension. I am doing a time course experiment so I would like to do all TCA concentration at the same time. Is it okay to snap freeze my media after collection, then thaw and concentrate the samples at a later time?
Thaw and concentration of protein at a later stage is okay but prolong storage may shear the protein causing smear formation in sds-page prior to WB.
Use ultrafiltration. For small volumes, you can use a centrifugal ultrafiltration device. For larger volumes, you can use a stirred pressure cell. If the medium contains fetal bovine serum, however, the protein concentration is already pretty high.
Mohannad Hazzazi
You can do a trichloroacetic acid (TCA) precipitation. This is the protocol I used:
1) Make 100% TCA inside a fume hood.
Note: Dissolve 500g TCA in final 227 mL of deionized water to make 100% TCA. Add the water in small increments to dissolve the TCA.
2) Add 100% TCA (final 10%) to the sample and vortex it.
3) Incubate the sample for 30 min on ice.
4) Centrifuge for 5 min at 14,000 rpm at 4oC.
5) Remove the supernatant without disturbing the pellet.
6) Wash the pellet with 150 µL of cold (-20oC) Acetone to remove any residual TCA.
Note: No need to break the pellet.
7) Centrifuge 5 min at 4oC at highest speed.
8) Remove the supernatant without disturbing the pellet and let the remaining Acetone to evaporate for 5 min.
Note: Do not over-dry the pellet.
9) Resuspend pellets in SDS-PAGE sample buffer.
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