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_at_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

Protein of Interest


Other Proteins
Lyse




4
IgG Functional Domains
Fab antigen binding region (epitope-specific)
Fc constant Region (host- specific)
Protein A binding region
5
Epitopes

• Continuous amino acids
• Non-continuous

Not every antibody will work well in every
application (IP, western, immunohistochemistry,
far-western, etc)
Adapted from Harlow and Lane, 1999
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

Lyse
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??

• A Quantitate amount of protein in lysate
• vs.cell/tissue debris (Western Blot)

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 _at_ 4C
• 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 _at_ 4C
• Saturating amounts of antibody
• Deplete all soluble protein of interest in lysate

13
Choosing an antibody
Rabbit Goat Hamster
Mouse
,
Common Problems
High background
Cross reactions w/ unrelated antigens
(specificity)
Not every antibody will work well in every
application (IP, western, immunohistochemistry,
far-western, etc)
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 _at_ 4C
• 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 _at_ 4C

17
Step 5 Detection ? Western Blot

• Western Blot to determine efficiency of IP

OR Perform Functional Assay
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

IP Ab goat anti-PoI goat IgG
WB mouse anti- PoI
19
Critical Controls

• 2. Parallel IP using sample that lacks antigen
• Ex untransfected cells
• Ex tissue that doesnt express antigen (null
  mutant)

Transfection
IP Ab goat anti- PoI goat IgG
WB mouse anti- PoI
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

IP Ab goat anti- PoI goat IgG
WB mouse anti- PoI
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
Other Applications

• 3. ChIP Chromatin Imunoprecipitation

Slide K. Gerrish
23
Crosslinking Antibodies

• 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

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

26
Physical Properties of Commonly Used Detergents
Molecular weight (Da) CMC Detergent mp
(?C) Monomer Micelle (w/v) M Anionic SDS 206 2
88 18,000 0.23 8.0 x 10-3 Cholate 201 430 4,300 0
.60 1.4 x 10-2 Deoxycholate 175 432 4,200 0.21 5.
0 x 10-3 Nonionic Octyl glucoside 105 292 8,000
0.73 2.3 x 10-2 Digitonin 235 1,229 70,000 C
12E8 542 65,000 0.005 8.7 x 10-5 Lubrol
PX 582 64,000 0.006 1.0 x 10-4 Triton
X-100 650 90,000 0.021 3.0 x 10-4 Nonidet
P-40 603 90,000 0.017 3.0 x 10-4 Tween
80 1,310 76,000 0.002 1.2 x 10-5
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