I have cultured primary enterocytes with lipids. After then I collected the medium and cells' microsome lumenal proteins for NaBr gradient (d=1.006~1.10), and each fraction (1ml) was collected after centrifugation.
First, I took some aliquots and add SDS-sample bfr, and analyzed by 6% SDS-PAGE. I could see apoB48 signals both in cell lysate and conditional medium lands, but not in fraction lands. So I supposed that the proteins in fractions in too less.
Also I have tried TCA/DOC method to precipitate proteins. I got great WB signals of low MW lipoproteins (
You should perhaps try to replace the TCA/NaDOC precipitation by a methanol/chlorofom precipitation (Wessel D & Flgge UI 1984. Anal.Biochem 138 pp 141 143).
To 0.2 ml sample add 0.6 ml methanol and 0.2 ml chloroform
Vortex
Add 0.6 ml water
vortex 1 minute
centrifuge 5 min 13 000g 4°C
Discard upper phase without disturbing the interphase that contains the proteins (I actually leave a bit of the upper phase to be sure)
Add 0.6 ml methanol
vortex
centrifuge 5 min 13 000g 4°C
remove completely the supernatant
dry pellets (SpeedVac or patience)
Dissolve pellets in 1X Laemmli load buffer
For solving the concentration problem, I refer you to my article entitled: "The Study of Extracellular Protein Fractions of Probiotic Candidate Bacteria on Cancerous Cell Line". In this work we used three methods: DOC and TCA precipitation techniques, lyophilisation and dialysis using PEG. Also you can use Amicon tubes for protein concentration.
Dear Dr. Robbins: Because most of papers used TCA/DOC method for this kind of fraction, I haven't tried others before. I'll try meoh/chcl3 and other methods side by side to check. Thanks for detailed protocol.
Dear Dr. Abdolalizadeh: Did the salt concentration effect lyophilized efficiency?
You can try protein concentrator spin columns (e.g., Amicon, or Corning Spin-X, etc.). This will allow you to concentrate the protein as well as changing your buffer. The high NaBr concentration will mess up the ionic strength of the SDS sample buffer and mess up the shape of the bands in electrophoresis.
I concur with Dr. Robbins regarding the precipitation of the fractions using organic solvent, and add that you should also perform the same precipitation on a whole cell lysate (not fractionated). This will allow you to determine if the precipitation alters the downstream analysis of your proteins.
Regarding IP, you can dilute the fractions with water to reduce the salt concentration. You may need to incubate with the primary antibody for a longer time (2-4x), but it should still work. Again, you can test this by preparing a sample using whole cell lysate incubated with high [NaBr].
Thanks for all useful responses! Spin columns would be a little bit expansive because I have 48 samples once a time. I would try MeOH/ChCl3 and IP method with input and fraction groups side by side to check which is better.
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