Talk:Knight:Protein solubility

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Notes from discussion with Kathleen

Check solubility of protein

  • His tags can promote aggregation of protein and formation of inclusion bodies.
  • Proteins can be insoluble/aggregate, even without the His tag. Sometimes it's a function of overexpression as well. It's a good idea to check the solubility of each new protein you make. If things are insoluble, you may be able to get enough protein by just purifying the soluble portion, purifying inclusion bodies and "refolding", or altering the expression conditions to increase solubility. Alternatively, you may consider making a new construct (moving the positon of the His-tag, expressing/purifying your protein as a fusion protein, etc.).--Kathleen

Procedure

  1. Grow a 3mL culture.
  2. Take 1mL of culture and
    1. Pellet cells
    2. Resuspend in buffer with 8M urea. (50–100 µL--Kathleen)
    3. Add SDS loading buffer.
    4. Lyse by heating at 95°C for 10 mins.
    5. Spin 10 mins at high speed in microcentrifuge.
    6. Save 5-10 μL to run on a gel. (This is the total protein.)
  3. Take another 1mL aliquot of culture and
    1. Pellet cells
    2. Resuspend in 50–100 µL of a "native" buffer.
    3. Add lysozyme (Lysozyme is ~14 kDa, so make sure it won't run in the same spot on a gel as your protein! If it is going to be a problem, freeze-thaw only should work reasonably well for this test. It is hard to sonicate small volumes. You could also try a commercial "mild lysis" reagent, although people in our lab have had varied success with these.--Kathleen)
    4. Freeze thaw 2-3 times at -80°C
    5. Spin 10 mins at high speed in microcentrifuge.
    6. Save some supernatant to run on a gel. (This is the soluble fraction).
    7. Resuspend pellet in buffer with 8M urea. (50–100 µL)
    8. Save some resuspended pellet to load on a gel. (This is the insoluble fraction).

Note: the amount of material you load from the supernatant and pellet should add up to the total protein so that you are comparing equivalent amounts.

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