Immuoprecipitation (IP) using Dynabeads

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Immunoprecipitation (IP) using Dynabeads are more efficient and reliable.
It has several advantages over the Crosslink Immunoprecipitation method.

• Reduce protocol time as short to 30 minutes only instead of a 2 day experiment
  No need for time-consuming procedures e.g. preclearing of cell lysates, 1 minute spin down each time etc.
• Materials will not be lost from spun-down resins or excess surface during pipetting
• Reduce the comsumption of expensive antibodies
• Fast and gentle method that causes minimal physical stress to target proteins
• Able to work with concentrated proteins solutions; therefore dilution is not required
  

Binding of Antibody (Ab)

1. Completely resuspend Dynabeads by pipetting or rotating on a roller (5 min).
2. Transfer 50 µl Dynabeads to a tube, place on magnet and remove supernatant.
3. Remove tube from magnet and resuspend the Dynabeads in 200 µl Ab Binding &
   Washing Buffer containing your Ab of choice. (Typically 1 - 10 µg Ab, the optimal
   amount needed will depend on the individual Ab used).
4. Incubate 10 minutes with rotation at room temperature.
5. Place tube on magnet and remove supernatant.
6. Remove tube from magnet and wash the Dynabeads-Ab complex by resuspending
   in 200 µl Ab Binding & Washing Buffer.

Immunoprecipitation of Antigen (Ag)

1. Place tube on magnet and remove supernatant
2. Add your Ag-containing sample (typically 100 - 1,000 µl) to the Dynabeads-Ab
   complex and gently resuspend by pipetting.
3. Incubate 10 minutes at room temperature with rotation.
   Place tube on magnet, transfer supernatant to a clean tube.
4. Wash the Dynabeads-Ab-Ag complex 3 times, using 200 µl Washing Buffer for
   each wash. Mix gently by pipetting.
5. Resuspend the Dynabeads-Ab-Ag complex in 100 µl Washing Buffer and transfer
   the suspension to a clean tube. Place tube on magnet and remove supernatant.

Elution of Ab/Ag complex (alternatives A: denaturing or B: non-denaturing)

• A. Gently resuspend the Dynabeads-Ab-Ag complex in 20 µl Elution Buffer. Add 10 µl
  NuPAGE® LDS Sample Buffer / NuPAGE® Reducing Agent mix and incubate 10 minutes
  at 70°C. Place tube on magnet and load supernatant/sample onto a gel. (Alternatively,
  the Dynabeads-Ab-Ag complex can be resuspended in the SDS sample buffer of your
  choice and heated as per your standard protocol prior to gel loading.)
• B. Gently resuspend the Dynabeads-Ab-Ag complex in 20 µl Elution Buffer. Incubate
  2 minutes at room-temperature. Place tube on magnet and transfer supernatant/sample
  to a clean tube.

However although the kit is fast and easy to use, however the yield is relatively little and there is alot of "background noise" due to the presence of heavy and light chains from antibodies (co-elution due to the lack of crosslinking in this protocol, therefore there is antibody contamination).
Background noise - Antibody contamination that leads to the production of a heavy and light chain bands appearing on the blot, hence rendering it hard to determine which band is the protein band i.e. immunoprecipitated.

I am currently trying to optimize this protocol by trying out preclearing of lysate (prevent nonspecific binding), incubating with sample and antibody for a longer period of time (to enhance yield) and crosslinking using DSS to prevent co-elution of antibodies; so as to produce better results in future.

Li Yinliang Alex
TG02 0704894E
Group 8
3 August 2009