I work primarily with S. cerevisiae and commonly perform western blotting on total protein lysates. Our lab uses a fairly standard TCA/Bead lysis method (https://www.med.unc.edu/pharm/dohlmanlab/resources/lab-methods/tca/) for extraction of total protein from cells. I base the re-suspension volume after lysis on OD600 (10 µL SDS Loading Buffer/10^7 cells), however, I am having difficulties getting optimal loading using this method (sometimes the loading is fine, whereas other times there is a large variation between samples of similar OD600). Does anyone else use this method and have tips for troubleshooting or can recommend an alternative method?
It is likely that the yield of extracted proteins is mostly determined by the efficiency of mechanical lysis by bead beating, the role of TCA being to denature any protease present in the extract before it has the opportunity to degrade other proteins. In turn, the efficiency of bead beating will depend on hydrodynamic parameters such as the volume of liquid, the volume of beads, the shape of the vessel, or the frequency of agitation. Careful control of these parameters is therefore important to achieve optimal extraction.
Another problem may be poor solubilisation of the TCA-precipitated proteins due to excess residual TCA present in the pellet. Make sure to suck off any liquid remaining above the pellet before adding the ré suspension buffer.
Hi Kedric, I have used a homogenizer and standard mortar and pestle with liquid Nitrogen to lyse the tissues and I cannot comment on the bead beating procedure. I used to get smeared bands of protein on the gel so I diluted the protein before running the protein on the gel. Maybe dilute the protein and check the OD value.
Also, you can check the concentration of the proteins to run and then dilute the final concentration to 10ug for each protein sample. Usually, we run between 5 ug to 20 ug protein samples in the gel.
Hello Kedric Milholland
May be you should try other methods of total protein quantification of your lysate. Usually, for Western Blot, Bradford or Bicinchoninic acid (BCA) assay is used for quantification of protein from the lysate. Using standard solutions of bovine serum albumin (BSA) you can produce a calibration curve of absorbance versus mass concentration. Assuming the analyte-proteins react in the same manner as the BSA standard, the unknown concentration can be determined. Then you can load 10ug - 20 ug of the total protein on the gel.
Best.
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