The difference between immunomagnetic beads and agarose beads for immunoprecipitation

(1) 24-48 hours after transfection

h

Cells can be harvested, add an appropriate amount of cell lysis buffer (containing protease inhibitors), and lyse on ice for 30 minutes.

Centrifuge the cell lysate at 4°C at maximum speed.

Centrifuge at maximum speed 30

After 30 minutes, take the supernatant;

(2) Take a small amount of lysate for Western

blot analysis, and add 1 μg of the corresponding antibody to the remaining lysate. into the cell lysate and incubate with slow shaking at 4°C overnight;

(3) Take 10 μl

protein

A

agarose Beads were washed with an appropriate amount of lysis buffer 3 times

, and centrifuged at 3,000

rpm for 3 minutes each time;

(4) Add 10 μl of pretreated

protein

A

agarose beads to the cell lysate incubated with the antibody overnight and incubate at 4°C with slow shaking for 2 -4h, couple the antibody to protein

A agarose beads;

(5) After immunoprecipitation reaction, incubate at 4°C

at 3,000< /p>

rpm

Centrifuge for 3 minutes

min, centrifuge the agarose beads to the bottom of the tube; carefully aspirate the supernatant, and use 1ml lysis buffer for the agarose beads Wash 3-4 times; finally add 15 μl of 2×SDS

sampling buffer and boil in boiling water for 5 minutes;

(6) SDS-PAGE,

Western

blotting or mass spectrometry analysis.

Issues to note:

(1) Mild lysis conditions are used for cell lysis, which cannot destroy all protein-protein interactions in the cells. Non-ionic denaturants (NP40) are mostly used. or Triton

X-100). Lysis conditions are different for each type of cell and are determined empirically. High-concentration denaturants (0.2% SDS) cannot be used, and various enzyme inhibitors, such as commercial cocktails, must be added to the cell lysate.

(2) Using clear antibodies, several antibodies can be used together.

(3) Use control antibody: