Immunoprecipitation Kit (Protein A) Protocol & Troubleshooting

Product No. 11719394001

Protocol

Cell Lysis and Sample Preparation

1. Wash cells/tissue at least twice with ice-cold PBS to remove any remaining serum proteins from the culture medium. For one immunoprecipitation reaction a sample volume of 1 to 3 ml is recommended. Using a microcentrifuge, a volume of 1 ml is optimal.

• Adherent cells should be washed by addition of PBS to the monolayer and disposal of the supernatant. Add lysis buffer cooled to +2 to +8 °C to the chilled, washed cell monolayers to achieve a concentration of 10⁶ to 10⁷ cells/ml. Scrape the cells to one side of the dish using a suitable device.
• Cells in suspension should be washed with PBS by centrifugation and resuspension of the pellet. Remove supernatant after the last wash. Resuspend the cell pellet in lysis buffer cooled to +2 to +8 °C to achieve a concentration of 10⁶ to 10⁷ cells/ml and transfer to an appropriate homogenizing device.
• Solid tissue should be washed by addition of PBS and disposal of the supernatant. Add lysis buffer to the sample to achieve a concentration of 5 to 20 mg tissue/ml.

2. Transfer sample to a Dounce homogenizer, pre-chilled on ice or any other type of micro-homogenizer. Be aware that the homogenization procedure might be critical for the functional integrity of the target antigen. Using a type B pestle, homogenize by repeated strokes (approx. 10).

3. Centrifuge homogenized suspension at 12,000 × g, 10 minutes, +2 to +8 °C in a table-top microfuge to remove debris. Alternatively, to prepare a high speed supernatant, centrifuge at 100,000 × g, 45 minutes, +2 to +8 °C.

4. Separate the supernatant and transfer to a microfuge tube (optimal volume 1 ml).

Preclearing with Protein A Agarose

To reduce background caused by non-specific adsorption of irrelevant cellular proteins to Protein A Agarose, a preclearing step is recommended.

5. Add 50 μL of homogeneous Protein A Agarose suspension (25 μL bed volume) to 1 to 3 ml sample and incubate at +2 to +8 °C for at least three hours or overnight on a rocking platform.

6. Pellet beads by gravity sedimentation or centrifugation at 12,000 × g for 20 s in a microfuge. Transfer supernatants to fresh tubes.

Immunoprecipitation of the Target Protein

7. Add an appropriate amount of the specific antibody and gently rock at +2 to +8 °C for one hour.

8. Add 50 μL of the homogeneous Protein A Agarose suspension to the mixture and incubate at +2 to +8 °C for at least three hours or overnight on a rocking platform.

9. Collect complexes by gravity sedimentation or centrifugation at 12,000 × g for 20 seconds in a microfuge.

10. Remove supernatant carefully, add 1 ml of wash buffer 1, resuspend the beads and incubate at +2 to +8 °C for 20 minutes on a rocking platform.

11. Repeat steps 9 and 10.

12. Collect complexes as described in step 9, remove supernatant, resuspend pellet in 1 ml of wash buffer 2, incubate at +2 to +8 °C for 20 minutes on a rocking platform, pellet the beads again and remove supernatant.

13. Repeat step 12.

14. Add 1 ml of wash buffer 3 to the pellet, resuspend, incubate at +2 to +8 °C for 20 minutes on a rocking platform, pellet the beads again and remove supernatant.

15. Remove the last traces of the final wash from the agarose pellet and from the walls and lid of the microfuge tube.

Gel Electrophoresis

The immunoprecipitated proteins can be separated by any type of one- or two-dimensional electrophoresis system providing sufficient protein resolution. For a detailed protocol for SDS-polyacrylamide gel electrophoresis or two-dimensional electrophoresis, please refer to one of the standard textbooks or to manuals from manufacturers of electrophoresis equipment.

1. Add 25 to 75 μL of gel-loading buffer to the immunoprecipitate.
2. Denature proteins by heating to 100 °C for 3 minutes. Remove Protein A Agarose by centrifugation at 12,000 × g for 20 seconds at +15 to +25 °C in a microfuge. Transfer supernatant to a fresh tube.
3. Analyze an aliquot by SDS-polyacrylamide gel electrophoresis.

Western Blotting

After electrophoresis, blot the gel onto a nitrocellulose or PVDF membrane using a standard Western blot protocol. To avoid damage or contamination of the membrane, always wear gloves when handling.

1. Hydrophobic membranes such as PVDF must be moisten with methanol for a few seconds and soaked with transfer buffer for at least 5 minutes. Nitrocellulose should be briefly soaked in water and then for at least 5 minutes in transfer buffer.
2. It is essential to thoroughly equilibrate the gel in transfer buffer for 5 to 10 minutes prior to transfer.
3. Blot according to standard protocols.
4. The blot can be stored dry for several months in a refrigerator if necessary, but must be re-wetted before starting immunodetection. PVDF membranes should be re-wetted in methanol or in 5% Tween 20 (v/v) solution.

Troubleshooting

No Signal

Sample preparation immunoprecipitation

• To reduce risk of antigen degradation during sample preparation, include additional specific protease inhibitors.
• Increase the concentration of the primary antibody up to 5 μg/ml.
• For low affinity antibodies, use washing buffers with lower stringency (e.g., 150 mM NaCl, no detergent).
• Check the affinity of the primary antibody to Protein A Agarose. If the affinity of the primary antibody turns out to be low, change to an appropriate matrix.

Detection

• Check if protein has been transferred properly to the membrane during blotting.-->If the transfer was not efficient, especially with high molecular weight proteins, change the transfer conditions (prolong the transfer time, increase current or change to alternative transfer buffers.
• High molecular weight bands are missing-->increase blotting time or change the transfer buffer.
• Check the enzyme activity of the secondary antibody conjugate. Dot different dilutions of enzyme-conjugate onto a blotting membrane and detect directly. If no signal appears, use fresh enzyme-conjugate and test in the same way. If still no signal appears, check the detection reagent.

Weak Signals

Sample preparation and immunoprecipitation

• To reduce the risk of antigen degradation during sample preparation include additional specific protease inhibitors.
• Optimize the concentrations of primary antibody.
• Prolong the incubation time with primary antibody to several hours at +2 to +8 °C.
• Prolong the incubation time with Protein A Agarose (overnight).
• Shorten the washing times. Use washing buffers with lower stringency (150 mM NaCl, no detergent).

Detection

• Increase the amount of protein applied to the gel.
• Check for efficient blotting.
• Prolong the detection time.

High Background

Sample preparation and immunoprecipitation

• Samples with high background may need several rounds of preabsorption to remove all the proteins binding nonspecifically to protein A.
• Increase the washing time of the antibody-Protein A Agarose complex after immunoprecipitation. Increase the stringency of washing conditions.
• Increased levels of background signals on the blot might be caused by the nonspecific trapping of proteins during centrifugation of Protein A Agarose/antigen complexes. This can be avoided by gravity-sedimentation of the complexes instead of centrifugation.

Detection

• Use clean equipment, freshly prepared buffers and new membranes.
• Dilute the protein concentration in the sample.
• Avoid touching the membranes. Use gloves and blunt-ended forceps with non-serrated tips.

Nonspecific Bands

Preclear the sample up to three times with Protein A Agarose prior immunoprecipitation steps in order to remove all the proteins binding non-specifically to protein A/G . If the serum immunoglobulins cannot be entirely removed during Protein A Agarose preclearing, serum-free cell culture conditions prior to cell lysis are recommended.
Alternatively, Protein A Agarose can be preloaded with the desired amount of specific antibody and the remaining protein A binding sites can be blocked with nonspecific control antibodies or serum.