Procedure

Solutions and Buffers Required: -

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

• 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.
  
• X microliter of Sepharose Beads Mixture is added to Cell Lysate. All the steps have to be done on ice till now.
  
• The mixture of Lysate and Beads is kept in a Rotary Agitator for 4 hours in 4 degrees.
  
• After the Incubation Time is over, the mixture is centrifuged, and the precipitate is washed with Lysis Buffer for three times.
  
• Wash the mixture with 100 microliters of Saline Solution
  
• 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.
  
• 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
  
• 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.

Protocol I: Lysis of Cell Monolayer (Adherent) Cultures

1. Carefully remove the culture medium from confluent cells.
2. Wash the cells once with PBS (phosphate-buffered saline).
3. Add ice-cold lysis/wash buffer to the cells and incubate on ice for 5 minutes with periodic mixing.
4. Transfer the lysate to a microcentrifuge tube and centrifuge at high speed to pellet the cell debris.
5. Transfer the supernatant to a new tube for protein concentration determination and further analysis.

Manual Immunoprecipitation

Note: To ensure bead homogeneity, mix the vial thoroughly by repeated inversion, gentle vortexing, or using a rotating platform.

1. Place an appropriate amount of Protein A/G magnetic beads into a microcentrifuge tube.
2. Add lysis/wash buffer to the beads and gently vortex to mix.
3. Place the tube into a magnetic stand to collect the beads against the side of the tube. Remove and discard the supernatant.
4. Add more lysis/wash buffer to the tube. Invert the tube several times or gently vortex to mix, then collect the beads with the magnetic stand. Remove and discard the supernatant.
5. Add the antigen sample/antibody mixture to the tube containing pre-washed magnetic beads and incubate at room temperature for 1 hour with mixing.
6. Collect the beads with a magnetic stand, remove the unbound sample, and save them for analysis.
7. Add lysis/wash buffer to the tube and gently mix. Collect the beads and discard the supernatant. Repeat this wash step twice.
8. Add ultra-pure water to the tube and gently mix. Collect the beads on a magnetic stand and discard the supernatant.
9. Low-pH Elution: Add elution buffer to the tube and incubate at room temperature with mixing for 10 minutes. Magnetically separate the beads and save the supernatant containing the target antigen. To neutralize the low pH, add a neutralization buffer to the eluate.

SDS-PAGE

1. Prepare the gel according to the laboratory instructions.
2. Mix the protein samples with the sample buffer and heat at 95°C for 5 minutes.
3. Load the samples and molecular weight marker into the wells of the gel.
4. Run the gel at a constant voltage until the dye front reaches the bottom of the gel.
5. Carefully remove the gel from the apparatus and proceed with the desired staining or transfer procedure for further analysis.