Griffin: Ultimate Immunoprecipitation Guide

If you are planning on performing an IP experiment, then I highly recommend you run the western blot first 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.

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Common RIPA components

PBS..........................................................Salt prevents non-specific protein aggregation Tris-HCl..................................................Buffering agent prevents protein denturation 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

Protease Inhibitors...................................Prevents protein degradation. You can make your own, or several vendors have convenient crushable pills that form a protease inhibitor cocktail solution. There are several;

Phosphatase Inhibitor..............................Prevents phsophatase activity.

NaVO4 (Sodium Orthovanadate) NaF (Sodium Fluoride)

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RIPA RECIPES

Standard Lysis Buffer I

RIPA buffer: 1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS. This may be made in large volumes. Add inhibitors fresh at time of use from the following stock solutions:

1) 10 mg/ml PMSF 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) 100 mM sodium orthovanadate in frozen aliquots (add at 10 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

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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).

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.

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.

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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.