Griffin: Ultimate Immunoprecipitation Guide

If you are planning on performing an IP experiment, then consider to run a preliminary western blot first in oder 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.

Common RIPA components

PBS : Salt prevents non-specific protein aggregation

Tris-HCl : 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.

RIPA Buffer Recipes

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

Lysis Buffer I' 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, PMSF, Aprotinin, 100 mM Na3VO4

1) 10 mg/ml PMSF (200 mM) in isopropanol (add at 10 µl/ml RIPA) 2) Aprotinin

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.

3) 200 mM sodium orthovanadate in frozen aliquots (add at 5 µl/ml RIPA)

Sodium orthovanadate should be activated for maximal inhibition of protein phosphotyrosyl-phosphatases .

-Prepare a 200 mM solution of sodium orthovanadate. -Adjust the pH to 10.0 using either 1N NaOH or 1N HCl. The starting pH of the sodium orthovanadate solution may vary with lots of the chemical. At pH 10.0 the solution will be yellow. -Boil the solution until it turns colorless (approximately 10 minutes). Cool to room temperature. -Readjust the pH to 10.0 and repeat steps 3 and 4 until the solution remains colorless and the pH stabilizes at 10.0. -Store the activated sodium orthovanadate as aliquots at -20°C. -This procedure depolymerizes the vanadate, converting it into a more potent inhibitor of protein tyrosine phosphatases. Please note that adding DTT rapidly inactivates sodium orthovanadate. Reference: Gordon, J.: Methods Enzymol. (1991) 201, 477-482 21:26, 2 September 2008 (EDT)21:26, 2 September 2008 (EDT)Korey Griffin 21:26, 2 September 2008 (EDT)

Lysis Buffer II An SDS free lysis buffer to consider with Co-IP approaches

150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% Nonidet P-40, 0.25% Sodium Deoxycholate, 1 mM EGTA, 1mM PMSF, 1 ug/ml of Aprotinin, Leupeptin, Pepstatin, 1 mM Na3VO4, 1mM NaF

Lysate Preparation

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

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

II) Add 0.8 ml of ice cold fresh RIPA buffer to the 100 mm cell culture plates.

III) 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.

IV) 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.

V) 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.

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

VII) 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

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-protein 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.