IGP Methodology: Immunoprecipitation

1. IGP Methodology: Immunoprecipitation A.J. Robison Colbran and Winder Labs Dept. of Molecular Physiology & Biophysics Nov. 1, 2005 a.j.robison@vanderbilt.edu

2. Immunoprecipitation Purpose: to isolate native protein from cellular/tissue environment Use antibody attached to sedimentable matrix Up to 10,000 – fold purification Uses: Protein – protein interactions Protein – DNA interactions Detection of post-translational modifications Determine rate of protein degradation/synthesis Enzymatic activity assays Protein detection (concentrates protein)

3. Immunoprecipitation - Basic Steps: Lysing of cells/homogenization of tissue Preclearing of lysate Addition of antibody to lysate Precipitation of Ab-antigen complex Detection

4. IgG Functional Domains

5. Epitopes Continuous amino acids: Non-continuous:

6. Step 1: Lyse Cells/Homogenize Tissue Goal: To use gentlest conditions possible to solubilize protein and maintain: shape (antibody recognition) activity/function Protein-protein (DNA) interactions

7. Step 1: Lyse Cells/Homogenize Tissue Purpose: to solubilize proteins and make accessible for antibody binding Characteristics to Consider about Your Protein of Interest: 1. Expression profile (abundance) 2. Solubility (membrane vs. cytosolic vs. nuclear) 3. Activity/function 4. Molecular weight 5. Availability of specific antibodies 6. Tagged protein-binding/activity/function 7. Sensitivity to mechanical disruption

8. Step 1: Lyse Cells/Homogenize Tissue Factors to consider in a lysis buffer: Salt concentration Type/ammount of detergent Presence of divalent cations pH Protease inhibitors Two common lysis buffers for IP: NP-40 buffer RIPA buffer (more harsh) Harsh lysis buffers: may denature / alter activity

9. Lysis Buffers Ionic vs. Nonionic detergents: -Nonionic: generally less denaturing -Use to solubilize membrane proteins AND maintain protein-protein interactions -Ionic: generally more denaturing -Use for difficult-to-solubilize proteins; may disrupt protein-protein interactions NP-40 lysis buffer: 150 mM NaCl 1.0% NP-40 (non-ionic detergent) [or Triton-X-100] 50 mM Tris, pH 8.0 RIPA buffer: more harsh than NP-40/Triton Can break apart protein-protein interactions/denature proteins disrupt membranes (for membrane protein extraction) 150 mM NaCl 1.0% NP-40 (non-ionic detergent) 0.5% sodium deoxycholate (ionic detergent) 0.1% SDS (ionic detergent) 50 mM Tris, pH 8.0

10. Step 1: Lyse Cells / Homogenize Tissue Q: How to determine lysis efficiency??

11. Step 2: Preclear Lysate Purpose: to remove proteins that may non-specifically bind to protein A/G or beads reduces background in later steps 1hr rotating @ 4°C Secondary reagents: (use wide-mouth tips) Protein A – agarose beads bacterial Fc receptors from Staphylococcus aureus Protein G – agarose beads bacterial Fc receptors from group G streptococci ** Wash beads separately in lysis buffer prior to use

12. Step 3: Add Antibody to Lysate Purpose: to bind all of your soluble protein of interest using specific antibodies Generally ~ 1-5 mg antibody per tube At least 1 hour rotating @ 4°C Saturating amounts of antibody Deplete all soluble protein of interest in lysate

14. Step 4: Precipitate Antibody-Antigen Complex Purpose: to precipitate all of the protein-bound antibody Saturating amounts of protein A/G agarose 1 hour rotating @ 4°C Binding capacity ~ 5-30 mg IgG/ml beads

15. Protein A vs. Protein G

16. Step 4: Precipitate Antibody-Antigen Complex Wash precipitate: remove proteins that have non-specifically bound to complex and to bottom of tube Decreases background/transiently-bound proteins Decreases non-specific proteins detected At least 3 x 5 min. washes (1 ml ea.) in IP buffer @ 4°C

17. Step 5: Detection ? Western Blot Western Blot: to determine efficiency of IP

18. Critical Controls: 1. Parallel IP with related Antibody Antibody from same species and subclass **Ideal antibody = preimmune serum Ex: Goat IgG as control for goat anti-PoI

19. Critical Controls: 2. Parallel IP using sample that lacks antigen Ex: untransfected cells Ex: tissue that doesn’t express antigen (null mutant)

20. Critical Controls: 3. Parallel IP: preadsorb antibody to antigen Usually an antigenic peptide Should prevent antibody binding to protein of interest in lysate Must use saturating amounts of antigen in preincubation

21. Other Applications: 1. Pulldown of tagged proteins • Glutathione agarose for GST-protein pulldown Express exogenous fusion protein in cells 2. Co-IP = Co-immunoprecipitation: • Pulls down proteins that are bound to PoI **Disadvantages/caveats

22. 3. ChIP = Chromatin Imunoprecipitation: Other Applications:

23. Protein A/G sepharose Prior to addition of antibody (step 3) Advantages: ? unbound Ab in lysate = ? antigen recovery Can now use same antibody (same species) for IP and western detection Otherwise, IgG can mask/distort proteins on gel: Crosslinking Antibodies:

24. Other Resources: **Using Antibodies: A Laboratory Manual Ed Harlow & David Lane Cold Spring Harbor Press, 1999 A Guide to the Properties and Uses of Detergents in Biology and Biochemistry Calbiochem Biochemicals, 1988 Company catalogs/product literature/websites Sigma-Aldrich, Upstate, Roche **Lab Protocols Current Protocols Online: Chapter 7, unit 7.2

25. The following slides are supplemental, and will not be discussed in class

27. General TIPS: Mix the protein A/G agarose slurry (50/50) well prior to use When adding agarose slurry solution: use wide mouth tip When removing supernatant, use smallest diameter pipet tip possible Lysate: generally 1 mg/ml protein for best results Crosslink antibody to beads