User:Reshma P. Shetty/Scratchpad

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(Notes from chat with Kathleen)
Line 74: Line 74:
===Check solubility of protein===
===Check solubility of protein===
*His tags can promote aggregation of protein and formation of inclusion bodies.
*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.).--[[User:Kathmc|Kathleen]]
====Procedure====
====Procedure====
Line 79: Line 80:
#Take 1mL of culture and
#Take 1mL of culture and
##Pellet cells
##Pellet cells
-
##Resuspend in buffer with 8M urea.
+
##Resuspend in buffer with 8M urea. (50–100 µL--[[User:Kathmc|Kathleen]])
##Add SDS loading buffer.
##Add SDS loading buffer.
##Lyse by heating at 95°C for 10 mins.
##Lyse by heating at 95°C for 10 mins.
Line 86: Line 87:
#Take another 1mL aliquot of culture and
#Take another 1mL aliquot of culture and
##Pellet cells
##Pellet cells
-
##Add lysozyme
+
##Resuspend in 50–100 µL of a "native" buffer.
 +
##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.--[[User:Kathmc|Kathleen]])
##Freeze thaw 2-3 times at -80°C
##Freeze thaw 2-3 times at -80°C
##Spin 10 mins at high speed in microcentrifuge.
##Spin 10 mins at high speed in microcentrifuge.
##Save some supernatant to run on a gel. (This is the soluble fraction).
##Save some supernatant to run on a gel. (This is the soluble fraction).
-
##Resuspend pellet in buffer with 8M urea.
+
##Resuspend pellet in buffer with 8M urea. (50–100 µL)
##Save some resuspended pellet to load on a gel.  (This is the insoluble fraction).
##Save some resuspended pellet to load on a gel.  (This is the insoluble fraction).

Revision as of 22:01, 27 September 2006

Random notes to keep track of

Contents

Gel shift assays

Chromosomal integration

  1. Haldimann A and Wanner BL. . pmid:11591683. PubMed HubMed [Haldimann-JBacteriol-2001]
  2. Platt R, Drescher C, Park SK, and Phillips GJ. . pmid:10610816. PubMed HubMed [Platt-Plasmid-2000]
  3. Katz L, Brown DP, and Donadio S. . pmid:2046656. PubMed HubMed [Katz-MolGenGenet-1991]
  4. Hasan N, Koob M, and Szybalski W. . pmid:7959062. PubMed HubMed [Hasan-Gene-1994]
  5. Diederich L, Rasmussen LJ, and Messer W. . pmid:1387714. PubMed HubMed [Diederich-Plasmid-1992]
  6. Le Borgne S, Bolívar F, and Gosset G. . pmid:15269421. PubMed HubMed [LeBorgne-MethodsMolBiol-1994]
All Medline abstracts: PubMed HubMed

Protein purification

Notes from the following book ...

  1. Robert K. Scopes. Protein purification. New York: Springer-Verlag, 1994. isbn:0387940723. [Scopes]

Chromatography

  • even packing and constant, even flow through the column is key to good results

Concentration

Precipitation

  • ammonium sulfate or sometimes acetone is used to precipitate protein and resuspend in a smaller volume
  • must remove precipitant
  • precipitation usually only works for protein solutions with concentration > 1mg/mL.
    • lower concentrations eithr don't precipitate or denature

Adsorption to an ion exchanger

  • good for dilute solutions

Dialysis

  • semi-permeable membrane removes water
  • ultrafiltration
    • very fast and effective on dilute solutions
  • centrifugal filtration
    • low volumes

Gel filtration

  • at least 10-15% loss of protein can be expected
  • can use centrifugation for small volumes[8]

Osmotic removal

  • sample is placed in dialysis tubing and immersed in solution or powder like PEG
  • be careful not to contaminate protein solution with polymer solution

Buffer exchange

Dialysis

  • often need multiple dialysis steps
  • proteolytic degradation may occur
  • slow

Gel filtration

  • preequilibrate column with buffer that the protein is to be exchanged into
  • rapid "desalting" occurs in one pass if the sample volume if < 1/5 column volume
  • small columns should be packed with finer grade beads to retain optimal resolution
  • rapid desalting of small samples with no dilution can be achieved by centrifugal method. Column of Sephadex G-25 is used inside a benchtop centrifuge.
  • sample should not have too much salt or protein for optimum separation
  • protein concentration should be < 30 mg/mL
  • doesn't matter what the column is washed with since protein travels before solvent front
  • final volume may be larger than initial volume before desalting
  • for buffer exchange, if protein concentration is low and volume large, "salt out" first and dissolve in a small volume of buffer and then use a relatively small desalting column

References

  1. Saul A and Don M. . pmid:6204553. PubMed HubMed [Saul-AnalBiochem-1984]
  2. Helmerhorst E and Stokes GB. . pmid:6247935. PubMed HubMed [Helmerhorst-AnalBiochem-1980]
All Medline abstracts: PubMed HubMed

Notes from chat with Kathleen

Thanks Kathleen! Note that any errors below are mine.

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.

General issues with protein purifications

  • Even when doing denaturing purifications, add 10mM imidazole to solutions to help with washing out non His tagged proteins.
  • May want to add small amounts of EDTA to the eluant to chelate heavy metals like Ni. Heavy metals can catalyze oxidation reactions that destroy your protein. However, the zinc content needs to account for the presence of EDTA.
  • Start with 1L of protein and do a larger scale prep. Thus, if the protein is crashing out of solution upon buffer exchange, you can see it very easily. With small volumes, it can be hard to see. If that happens, just start playing with the pH and salt content.
  • For buffer exchange, the Sauer lab generally does either dialysis or gravity flow gel filtration (much faster). However they are usually working with much larger volumes ... 1L culture eluted in 2.5-3 mL. They then run this through a PD10 column from Amersham. Or else 0.5-1mL through a NAPS column. There are also columns for 20-100μL.

Native purifications

  • Sauer lab uses a slightly higher salt concentration than what the Qiagen manual calls for because supposedly the wash is improved somewhat. This was done with a DNA binding protein.
  • Can lyse cells by doing repeated freeze-thaw cycles at -80$deg;C or sonication also works.
  • Can consider using a SlyD knockout strain. SlyD is a 20-25 kDa protein that has several histidines near each other and can often contaminate Ni column purifications.

Refolding a protein on the column

  1. Lyse cells in denaturing buffer supplemented with 10 mM imidazole
  2. Run through column
  3. Wash with "native" wash buffer (contains 20mM imidazole)
    • The protein should refold on the column.
  4. Elute in "native" elution solution.

Centrifugal filtrations

  • Even though they are not supposed to, sometimes these columns can end up concentrating your buffers. This may explain why my dye front runs oddly when loading samples from the microcon. The salt content has been increased.
  • These columns tend to bind an absolute amount of protein, not a fraction of what you send through it. Hence, larger scale preps tend to be preferable.
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