Myers:ProteinComplexPurification

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Contents

Overview

Purification of Proteins Complexes with in vivo chemical crosslinking

Materials

NETN (50 mM HEPES-HCl [pH 7.5], 150 mM NaCl, 0.5-1% Igepal, and 1%glycerol optional)

Protease and phosphatase inhibitors

DSP [MW 404.42] (Pierce): Prepare the DSP by adding X mg so that the final concentration of DSP is 1.5mM when added to NETN buffer. Prepare the DSP in Y volume of DMSO so that the total concentration of DMSO is 1% when added to the NETN buffer. For example add 0.606mg of DSP per 1ml of NETN prepared in 10ul of DMSO. For ease I suggest preparing 3.033mg in 50ul of DMSO and using 10ul per 1ml. DSP can be used down to 0.2mM final concentration. Be sure to reverse crosslinks by incubating 60°C for 30min in sample/loading buffer with 50mM DTT. Optional: DTTSP [MW 608.51] (Pierce) can be used instead of DSP. DTTSP is water soluble and is used at a lower concentration i.e 0.01-1mM. For ease in conversion 0.6mg/ml gives 1mM. I suggest using 0.1mM and this is added directly to lysis buffer.

1M Tris-HCl pH7.5

5M LiCl

Protein A/G suspension (Pierce) + immunoprecipitation proficient antibody

Flag-Resin (Sigma – I prefer EZ-view)

3X Flag peptide (Sigma) - Optional

HA-Resin (Sigma)

HA peptide (Sigma)- Optional

Cell Culture: Culture cells without allowing the cells to reach confluency greater than 95%. Typically 3-10 15cm plates are sufficient for abundant or ectopically expressed proteins. Harvest cells by trypsinization and washed by centrifugation @ 1000rpm. Wash the cells once in 1X PBS and flash freeze in liquid nitrogen.

Procedure

Lysis and in vivo crosslinking:

  1. Lysed cell pellets on ice in 20-100 pellet volumes of NETN-XL buffer (50 mM HEPES-HCl [pH 7.5], 150 mM NaCl, 1% Igepal, and 5%glycerol) containing protease inhibitors (1mM phenylmethyl-sulfonyl fluoride, 1ug/ml leupeptin, 1ug/ml pepstatin, and 1ug/ml aprotinin-200X stock solution), 1x MSRC phosphatase inhibitor cocktail (250X stock solution), 1X Sodium orthovandate (200X stock solution), and 1.5mM DSP (pierce) in 1% lysis volume DMSO (see Materials section).
  2. Sonicate cell lysate 2x with 15 sec pulses.
  3. Incubate the lysate for 25min on ice with occasional inversion.
  4. Quench crosslinking by adding Tris-HCl pH7.5 to final concentration of 50mM using 1M Tris-HCl pH7.5. Use 5ul per 1ml lysis buffer.
  5. Clear lysates by centrifugation at 8000 rpm for 10 minutes.
  6. Determine protein concentration and dilute the lysate to 1-5mg/ml. Small scale immunoprecipitations use between 0.5 – 2mg/ml total protein. Large immunoprecipitations require ~10-fold increase in total protein.
  7. Aliquot ~190ug of lysate (-500ul) was and combined with LDS loading buffer and DTT (@ final concentration of 50 mM) and used as input.
  8. The remaining lysate (in 4.5mL) is transferred to clean tubes and incubated with antibody or with washed Flag or HA resin
  9. Antibody or resin is incubated with lysate at 4°C for 1.5hrs.
  10. The suspension is centrifuged @2000rpm (~0.5xg) for 2 min @4°C.
  11. Immunocomplexes are washed 3x with NETN (without crosslinker) buffer by inverting tube 15 times.
  12. For denaturing elution 2 resin volumes 2XLDS + 50mM DTT( DTT @ final concentration of 50mM) by heating for 20min at 60°C.
  13. Native elution is possible when using 1X Flag-tagged target protein by adding 300ug/ml 3X Flag peptide in 20mM Tris-HCl pH7.5 and 750mM LiCl. For 400ul of 3X Flag peptide solution use 8ul of 1M TrisHCl, 60ul of 5M LiCl, 25ul of 3X Flag peptide ( 5 mg/ml) and 307ul of Ultrapure H2O. This elution is in done twice and precipitated using TCA. I usually use 75-100ul of resin and elute using 200ul of Flag peptide with each elution for a total of 400ul.
  14. TCA precipitation is done by adding 1/5 volume of 100% TCA (100ul of 100% TCA to 400ul of elution).
  15. Incubate the TCA precipitation overnight on ice.
  16. Centrifuge max speed for 10min and remove all of the supernatant.
  17. Centrifuge again for 2min and remove all supernatant.
  18. Resuspend the pellet in 25ul of 200mM Tris-HCl pH8.5 and add 25ul of 2X LDS containing 100mM DTT.
  19. Incubate this 20min at 60°C.
    • always keep lysates on ice and do not introduce bubbles during sample preparation as the surface tension can denature proteins.

Notes

  1. Be sure to follow standards for protein handling and immunoprecipitation.
  1. For large scale IPs I use 10-15cm plates, lyse in 12.5-25ml of lysis buffer, use 75ul of packed resin for Flag and HA-tagged proteins, wash with 5-7ml of NETN, elute with 200ul of resin in each elution for a total elution volume volume of 400ul, precipitate with 100ul of 100% TCA,

Properties of DSP: Alternate names = Lomant’s reagent; Molecular formula = C14H16N2O8S2;Molecular weight = 404.42; Spacer arm length = 12.0 Å (8 atoms);CAS Number = 57757-57-0; Storage conditions = 4°C, protect from moisture, use only fresh solutions; Reactive groups = NHS esters, react with primary amines at pH 7.0-9.0


References

Relevant papers and books

  1. DSP http://www.piercenet.com/Objects/View.cfm?type=ProductFamily&ID=02030234 [For]
  2. Flag and HA http://www.sigmaaldrich.com/

    [For]

Contact

  • Jeremy Myers

--Jeremy S. Myers 11:33, 10 April 2008 (EDT) or instead, discuss this protocol.

Post questions or comments on my talk

http://openwetware.org/wiki/User_talk:Jeremy_S._Myers

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