Griffin: Ultimate Immunoprecipitation Guide

Serum levels of human Ig isotypes

If you are planning on performing an IP experiment, then consider to run a preliminary western blot first in order to get a feel for what the antibody is capable of binding.

RIPA (Radio ImmunoPreciptation Assay) buffer is a traditional name for an array of recipes that have found success over the years. Below are details that can contribute to optimizing your immunoprecipitation experiment. Lysis buffer components can and will influence the efficiency of the IP reaction. Detergent composition is a major factor for IP reactions. Adjusting salt concentration and detergent composition will influence the efficiency of the IP reaction. Membrane bound proteins (proteins based in lipid rafts), protein complexes, and protein charge can all influence the efficient yield of your gene product in the lysate prep.

The amount of protein present in cells will vary with cell type. 1 to 10 million cells can yield ~1mg of protein.

Common RIPA components

PBS : Salt prevents non-specific protein aggregation

Tris-HCl : Buffering agent prevents protein denaturation

NaCl : Buffering agent prevents protein denaturation

1% Nonidet P-40 or Igepal CA-630 : Non-ionic detergent to extract proteins, form lipid micelles

1% Triton X-100 : Non-ionic detergent to extract proteins, form lipid micelles - to use in place of Nonidet/Igepal

0.5% sodium deoxycholate : Ionic detergent to extract membrane protein and isolate lipids

0.1% SDS : Ionic detergent to extract membrane protein and isolate lipids

EGTA : Protease Inhibitor, Prevents protein degradation. You can make your own, or several vendors have convenient crushable pills that form a protease inhibitor cocktail solution.

Na3VO4 (Sodium Orthovanadate) : Tyrosine phosphatase Inhibitor; hydrostatic interference of active sights of phosphatases

NaF (Sodium Fluoride) : Serine/Threonine phosphatase Inhibitor; hydrostatic interference of active sights of phosphatases

Misc. Phosphatase Inhibitors: Phosphorylation/dephosphorylation of proteins influences the charge-charge relationships that proteins have with eachother in solution. Proteins undergo covalent attachment of a phosphoryl group (phosphorylation) typically at serine, threonine, or tyrosine residues. Phosphate groups are removeable via protein phosphatases. During the extraction of phosphorylated proteins from cell and tissue, preserving the phosphorylation states of total protein is a good technique.

Protease Inhibitor Cocktails

Tableted formulations containing water-soluble protease inhibitors are user-friendly and effective substitutes in lysis buffers. When using divalent/trivalent columns for enrichment of phosphoproteins, be sure to avoid EDTA/EGTA containing cocktails.

Examples:

• S8820: Sigma SIGMAFAST™ Protease Inhibitor Tablets
• 78429: PIERCE Halt Protease Inhibitor Cocktail (100X)
• 04693116001: Complete™ Roche Protease Inhibitor Cocktail Tablets
• sc-29131: Santa Cruz Biotechnology Inc.,Protease Inhibitor Cocktail Tablet, EDTA-free

Protein A/G/L Agarose

Binding Affinity Guide

Protein A & Protein G bind to most mammalian immunoglobulins primarily through their Fc regions. Protein L is a kappa light chain specific Ig-binding protein.

Protein A/G/L are common to covalently couple by cyanogen bromide to highly cross-linked 4% agarose beads. This type of matrix is stable in most aqueous buffers.

• Typical Ligand density: ~3 ug Protein/ul of bead
• Binding capacity: ~15-20 ug Ig/ul bead
• Bead structure: 4-6% cross-linked agarose
• Bead diameter: 40-170 um
• Temperature stability: 4-40 C

Protein A

Native protein A is a single chain (predicted 42 kDa, SDS-PAGE 46 kDa), glycoslyation-free, cell wall component produced in strains of Staphylococcus aureus. Protein A binds specifically to the Fc region of immunoglobulin molecules, including IgG. Protein A has four high-affinity (Ka = 108/mole) binding sites toward Fc region of IgG of several species (two sites can bind at a time). Protein A is heat-stable and retains conformation even after exposure to denaturing reagents such as 4 M urea, 4 M thiocyanate and 6 M guanidine hydrochloride.

Protein G

Native protein G is a bacterial cell wall protein from group G Streptococci that contains two immunoglobulin binding sites, an albumin binding site, and cell surface binding sites. Native Protein G. The recombinant form of Protein G (predicted 17-21 kDa; SDS-PAGE 31-34 kDa) contains only the two immunoglobulin binding sites to reduce nonspecific binding when purifying immunoglobulins.

Protein L

Native protein L is an kappa light chain specific Ig-binding protein that originates from the bacteria Peptostreptococcus magnus. Protein L binds Igs through interactions with the light chains. Because no part of the heavy chain is involved in the binding interaction, Protein L binds a wider range of Ig classes than Protein A or G. Protein L binds to representatives of all classes of Ig, including IgG, IgM, IgA, IgE and IgD. Single chain variable fragments (ScFv) and Fab fragments also bind to Protein L.

In humans and mice, kappa (k) light chains predominate. The remaining immunoglobulins have lambda (l) light chains. Furthermore, Protein L is effective in binding only certain subtypes of kappa light chains. For example, it binds human VkI, VkIII and VkIV subtypes but does not bind the VkII subtype. Binding of mouse immunoglobulins is restricted to those having VkI light chains.

Protein L matrix is suitable for purification of kappa light chain-containing monoclonal antibodies from ascites or culture. Protein L is useful for purification of VLk-containing monoclonal antibodies from culture supernatant because it does not bind bovine immunoglobilins, which can be present in media. Protein L does not interfere with the antigen-binding site of the antibody.

RIPA Buffer Recipes

There are 3 different RIPA style lysis buffers outlined below including a brief summary of their purpose.

These may be made in large volumes. Add inhibitors fresh at time of use from stock solutions

Lysis Buffer 1:

A tried and true lysis buffer for most signaling intermediates and soluble/cytosolic factors.

• 1x PBS
• 1% Nonidet P-40
• 0.5% sodium deoxycholate
• 0.1% SDS
• 100 mM Na3VO4 Sodium orthovanadate activation
• 10 mg/ml PMSF (200 mM) in isopropanol (add at 10 µl/ml RIPA)
• Protease inhibitor cocktail

Aprotinin activity is measured by KIU (KIU = Kallikrein Inhibitory Unit) Since the vial contains other components which makes the total dry. I recommend the following procedure to be used with this product: The normal working concentration range for aprotinin is either 0.5ug-2ug/ml (protein weight/volume) or 10 KIU-100 KIU/ml (units/volume). 1ug aprotinin/ml of RIPA buffer works well.

Lysis Buffer 2:

An SDS free lysis buffer to consider with Co-IP approaches. Used this one in graduate school to IP EGFR effectively.

• 150 mM NaCl
• 50 mM Tris-HCl pH 7.4
• 1% Nonidet P-40
• 0.25% Sodium Deoxycholate
• 1 mM EGTA
• 1mM PMSF
• Protease inhibitor cocktail
• 1 mM Na3VO4
• 1mM NaF

Lysis Buffer 3:

This recipe calls for Brij 35 which is a non-ionic detergent, great for dissociating membrane complexes and essentially much gentler than SDS. This is also a great lysis buffer for phospho-proteins.

• 10 mM KPO4 (phosphate buffer)
• 1 mM EDTA (chelate)
• 5 mM EGTA (chelate)
• 10 mM MgCl2 (chelate)
• 50 mM †-glycerophosphate (inhibits serine-threonine phosphotase activity)
• 0.5% NP-40 (stabilizer of proteins/enzymes)
• 0.1% Brij 35 (non-ionic detergent)
• 0.1% deoxycholic acid (non-ionic, non-denaturing detergent)
• 1 mM sodium orthovanadate (inhibits tyrosine phosphotase activity)
• Protease inhibitor cocktail

1. Roux PP, Richards SA, and Blenis J. Phosphorylation of p90 ribosomal S6 kinase (RSK) regulates extracellular signal-regulated kinase docking and RSK activity. Mol Cell Biol. 2003 Jul;23(14):4796-804. DOI:10.1128/MCB.23.14.4796-4804.2003 |

Adherent cell sample preparation

Below is a procedure for adherent cells (ie A431, A549, Hela, NIH3T3)

• Remove culture medium and rinse a subconfluent, 100 mm cell culture plate (80% confluent plate yields ~600-1000 ug protein total) with PBS at room temperature. The following steps should be performed on ice or at 4° C using fresh, ice cold buffers.

Optional: For monolayer cells, do a trypsin treatment to lift cells off the flasks prior to adding the RIPA buffer, instead of scraping the cells for a more gentle approach. If you are running a time course experiment, this is not feasible since the cells must be arrested and lysed immediately.

• Add 0.8 ml of ice cold fresh RIPA buffer to the 100 mm cell culture plates OR 0.5 ml per 5 x 10e6 cells/60 mm dish.

• Gently rock plates for 15 minutes at 4° C or let the plates set on ice. This step will allow the lysis buffer to act on the cells and will increase the total yield of soluble protein. Scrape the adherent cells with a cell scraper and then transfer the scraped lysate into a sterile microcentrifuge tube. Place the tube on ice.

Optional: wash the plate once with 0.2 ml of RIPA buffer and combine with first lysate. When running multiple plates this can be tedious and not necessary if enough attention is given to the initial harvest.

Optional: Add 10 µl of 10 mg/ml PMSF stock to the lysate. If a protease inhibtior cocktail is used fresh with the RIPA buffer, this is not necessary.

• Sonicate each sample on a 70% duty cycle or less by placing only the very tip of the pin into the vial, then slowly lowering it into the lysate until it foams completely and then stop. Alternatively, pass the lysate through a 21 gauge needle to shear the DNA & incubate 30–60 minutes on ice.

• Microcentrifuge cell lysates at 12,000xg for 15 minutes at 4°C.

• The supernatant fluid is the total cell lysate. Transfer the supernatant to a new microfuge tube and discard the pellet. Quantitate the protein amount by Bradford or BCA.

Suspension cell sample preparation

• Collect approximately 2.0 x 107 cells by low-speed centrifugation (e.g. 200xg) at room temperature for 5 minutes. Carefully remove culture medium.

• Wash the pellet with PBS at room temperature, and again collect by low-speed centrifugation. Carefully remove supernatant.

• Add 1.0 ml of ice cold RIPA buffer with freshly added (Protease Inhibitors) and/or (Phosphatase Inhibitors). Gently resuspend cells in RIPA buffer with a pipet and incubate on ice for 30 minutes.

• Further disrupt and homogenize cells by hydrodynamic shearing (21-gauge needle), dounce homogenization or sonication, taking care not to raise the temperature of the lysate. (Optional: Add 10 µl of 10 mg/ml PMSF stock; sc-3597) Incubate 30 minutes on ice.

• Transfer to microcentrifuge tube(s) and centrifuge at 12,000xg for 15 minutes at 4° C. The supernatant fluid is the total cell lysate. Transfer the supernatant to a new microfuge tube. This is your whole cell lysate. For increased protein recovery, resuspend the pellet in a small volume of RIPA, centrifuge and combine supernantants.

• The supernatant fluid is the total cell lysate. Transfer the supernatant to a new microfuge tube and discard the pellet. Quantitate the protein amount by Bradford or BCA.

Tissue extract sample preparation

There are a few approaches to optimizing protein yield from whole tissue extract. For whole animal studies, arresting the covalent modification state of the entire proteome is essential to obtaining accurate data about the treatment or phenotype effect on the tissue being extracted.

1) Immediately liquid nitrogen flash freeze tissue/organ then stored at -80 C

2) Immediately heat denature the organ from the sacrificed animal by microwave in a sealed container

• Weigh tissue and dice into very small pieces using a clean razor blade. Frozen tissue should be sliced very thinly and thawed in RIPA buffer containing (Protease Inhibitors) and/or (Phosphatase Inhibitors). Use 3 ml of ice cold RIPA buffer per gram of tissue.
• Further disrupt and homogenize tissue with a dounce homogenizer or a sonicator, maintaining temperature at 4° C throughout all procedures. (Optional: Add 30 µl of 10 mg/ml PMSF (sc-3597) stock per gram of tissue.) Incubate on ice for 30 minutes.
• Transfer to microcentrifuge tubes, centrifuge at 10,000xg for 10 minutes at 4° C. Remove supernatant and centrifuge again. The supernatant fluid is the total cell lysate. A longer centrifugation may be necessary to obtain a clear lysate.
• The supernatant fluid is the total cell lysate. Transfer the supernatant to a new microfuge tube and discard the pellet. Quantitate the protein amount by Bradford or BCA.

Immunoprecipitation Procedure

I) Incubate cell lysate (500-1000 ug) with (2-5 µg) primary antibody (optimal antibody concentration should be determined by titration) for 2 hours at 4°C.

II) Add 20 µl of appropriate agarose conjugate suspension (Protein A-Agarose, Protein G-Agarose, Protein A/G-Agarose or Protein L-Agarose).

Protein A-Agarose : specific binding to mouse IgG2a, IgG2b and IgA, rabbit polyclonal Abs, human IgG1, IgG2 and IgG4

Protein G-Agarose : specific binding to mouse IgG1, IgG2a, IgG2b, IgG3, rat IgG1, IgG2a, IgG2b and IgG2c, rabbit and goat polyclonal Abs, human IgG1, IgG2, IgG3 and IgG4

Protein L-Agarose : specific binding to mouse, rat and human IgG, mouse and human IgM, IgE and IgA proteins and scFv and Fab fragments

What is Protein G PLUS? Protein G PLUS is a genetically engineered form of streptococcal Protein G that has an increased capacity and has had the albumin binding site removed to reduce background.

III) Cap tubes and incubate at 4 C on a rocker platform or rotating device for 2 hour to overnight.

IV) Collect pellet by centrifugation at 2,500 rpm (approximately 1,000xg) for 5-10 seconds. A touch spin will work. With enough samples, gravity will pellet the beads as well.

V) Carefully aspirate and discard supernatant. The trick here is to slowly aspirate with the needle touching just the top of the liquid and slowly draw down so that the needle is pulling at the surface tension of the supernatant. This will ensure no loss of beads.

VI) Wash pellet 3 times with either RIPA buffer (more stringent) or PBS (less stringent), each time repeating centrifugation step above.

VII) After final wash, aspirate and resuspend pellet in 40 µl of 2x electrophoresis sample buffer. Or elute proteins with an appropriate antibody elution buffer.

VIII) Boil samples for 2 minutes. Load sample.

Related Recipes

Electrophoresis sample buffer (2x): Mix 1.0 ml glycerol, 0.5 ml 2-mercaptoethanol, 3.0 ml 10% SDS, 1.25 ml 1.0 M Tris-HCl, pH 6.7, and 1–2 mg bromophenol blue. Store frozen in small aliquots. Alternatively, make buffer without 2 -mercaptoethanol and store at room temperature. Add 2-mercatoethanol just before using.

Sample buffer formulation:

• 7 ml Tris·Cl/SDS, pH 6.8
• 3.0 ml glycerol (30% final)
• 1 g SDS (10% final)
• 0.93 g DTT (0.6 M final)
• 1.2 mg bromphenol blue (0.012% final)
• Add H2O to 10 ml
• Store in 0.5-ml aliquots at -70°C

References

Santa Cruz Biotechnology protocols