Procedure :

Solutions and Buffers Required: -

1. Lysis Buffer-Tris, NaCl, EDTA, Glycerol (Maintained at pH-7.4)
2. Saline Solution- NaCl
3. SDS PAGE Sample Buffer-Contains Tris HCl, DTT, SDS, Glycerol, Bromophenol blue. It is usually diluted 2X
4. Elution Buffer-IgG Elution Buffer or Glycine HCL.
5. Protease inhibitors
6. PBS Buffer

Preparation of Immune Complex-
The protein sample is prepared, and X microliter of Lysis Buffer is added (Typically in 10:1 ratio)
2-10 ug of purified antibody is added along with Cell Lysate in microcentrifuge tube
The mixture is diluted with 300-600 microliter of Lysis Buffer
It is incubated at 4 degrees for 1 hour to let the immune complex formed

1. Sepharose Beads are prepared. If it is a monoclonal antibody, protein G coupled Beads are preferably chosen, and if it is polyclonal antibody, protein A-coupled beads are used. Around 1ml PBS is added and then the mixture is centrifuged, the supernatant is removed and a buffer containing protease inhibitors (generally Lysis/Wash Buffer) is added to the precipitate and is ready to be used. Sometimes the beads can be washed twice before the buffer is added.
2. X microliter of Sepharose Beads Mixture is added to Cell Lysate. All the steps have to be done on ice till now.
3. The mixture of Lysate and Beads is kept in a Rotary Agitator for 4 hours in 4 degrees.
4. After the Incubation Time is over, the mixture is centrifuged, and the precipitate is washed with Lysis Buffer for three times.
5. Wash the mixture with 100 microliters of Saline Solution
6. Finally, X microliter of Loading Buffer is added, and the mixture is boiled at 100 degrees for 5 minutes to separate the beads and protein. Further, the centrifuged supernatant is stored.
7. Now the supernatant (containing isolated protein) can be eluted in two different elution buffers, depending on the use of the SDS sample elution buffer and the low pH elution buffer. i) If we want to elute in SDS sample elution buffer we can do it in 50 microliters of SDS Sample Elution Buffer, centrifuge, and collect the supernatant. ii) We can do in low pH elution buffer (50 microliters), centrifuge and collect the supernatant
8. The isolated proteins can undergo a western blot for further analysis.

*The Antibody Datasheet can be used to check the amount of Antibody concentration to be used.