Griffin: Ultimate Immunoprecipitation Guide

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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

Immunoprecipitation Procedure

Prepare tissue or cell culture samples.

Incubate 1 ml 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.

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

Cap tubes and incubate at 4 C on a rocker platform or rotating device for 1 hour to overnight.

Collect pellet by centrifugation at 2,500 rpm (approximately 1,000xg) for 5-10 seconds. A touch spin will work.

Carefully aspirate and discard supernatant.

Wash pellet 3 times with either RIPA buffer (sc-24948) (more stringent) or PBS (less stringent), each time repeating centrifugation step above.

After final wash, aspirate and resuspend pellet in 40 µl of 2x electrophoresis sample buffer (sc-24945).

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.