Purification of Proteins Complexes with in vivo chemical crosslinking
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
Protein A/G suspension (Pierce) + immunoprecipitation proficient antibody
Flag-Resin (Sigma – I prefer EZ-view)
3X Flag peptide (Sigma) - Optional
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.
Lysis and in vivo crosslinking:
- 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).
- Sonicate cell lysate 2x with 15 sec pulses.
- Incubate the lysate for 25min on ice with occasional inversion.
- 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.
- Clear lysates by centrifugation at 8000 rpm for 10 minutes.
- 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.
- Aliquot ~190ug of lysate (-500ul) was and combined with LDS loading buffer and DTT (@ final concentration of 50 mM) and used as input.
- The remaining lysate (in 4.5mL) is transferred to clean tubes and incubated with antibody or with washed Flag or HA resin
- Antibody or resin is incubated with lysate at 4°C for 1.5hrs.
- The suspension is centrifuged @2000rpm (~0.5xg) for 2 min @4°C.
- Immunocomplexes are washed 3x with NETN (without crosslinker) buffer by inverting tube 15 times.
- For denaturing elution 2 resin volumes 2XLDS + 50mM DTT( DTT @ final concentration of 50mM) by heating for 20min at 60°C.
- 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.
- TCA precipitation is done by adding 1/5 volume of 100% TCA (100ul of 100% TCA to 400ul of elution).
- Incubate the TCA precipitation overnight on ice.
- Centrifuge max speed for 10min and remove all of the supernatant.
- Centrifuge again for 2min and remove all supernatant.
- Resuspend the pellet in 25ul of 200mM Tris-HCl pH8.5 and add 25ul of 2X LDS containing 100mM DTT.
- 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.
- Be sure to follow standards for protein handling and immunoprecipitation.
- 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
Relevant papers and books
- DSP http://www.piercenet.com/Objects/View.cfm?type=ProductFamily&ID=02030234
- Flag and HA http://www.sigmaaldrich.com/
- Jeremy Myers