免疫沉淀實驗操作步驟及該選擇Protein A還是G？

所需器材：

①M(fèi)icrocentrifuge
②1.5 ml microcentrifuge tubes
③Rocker platform
④Scintillation counter
⑤GF/C Whatman filters
⑥X-ray film or phosphoimager equipment

溶液和試劑：

①M(fèi)onoclonal or polyclonal antibody
②PBSTDS: Mix 100 ml of 10×PBS with 10 ml of 100% TRITON X-100 , 5 g of sodium deoxycholate, and 1 g of sodium dodecyl sulfate; include a cocktail of protease inhibitors - 0.5 mg/ml of leupeptin , 1 mM EDTA, 1 mg/ml of pepstatin A, and 0.2 mM PMSF; add distilled water to 1L
③Immunoglobulin from normal animals
④10% TCA (trichloroacetic acid)
⑤Scintillation fluid
⑥Protein A-agaros or Protein G-agarose

該選擇Protein A-agaros還是Protein G-agarose呢？這要根據(jù)你選擇的抗體來定。比如，我要用的抗體是Mouse IgG2a，那么我選擇ProteinA或者G都是可以的，因為親和力大小一樣。

不同類型抗體與Protein A 或G親和力比較

Protein A/G Affinities for Various Monoclonal Antibodies
Species
Affinity for Protein A
Affinity for Protein G
Human IgG1
++++
++++
Human IgG2
++++
++++
Human IgG3
-
++++
Human IgG4
++++
++++
Rat IgG1
-
+
Rat IgG2a
-
++++
Rat IgG2b
-
++
Rat IgG2c
+
++
Mouse IgG1
+
++++
Mouse IgG2a
++++
++++
Mouse IgG2b
+++
+++
Mouse IgG3
++
+++

操作步驟：

NOTE: this procedure is written for the use of agarose beads as the precipitating reagent.

①Pre-clear the lysate or conditioned media before using it for immunoprecipitation by adding 1 mg of normal IgG from the appropriate species to the sample together with 20 ml of the agarose conjugate of choice; this will remove proteins that precipitate non-specifically and would thus contaminate the immunoprecipitate.

②Incubate for 1 hour at 4℃; centrifuge the sample at 1500 rpm to pellet the agarose; carefully collect and save the supernatant fluid and use for specific immunoprecipitation.

③For radiolabeled samples:
A. Determine the TCA precipitable counts in the radiolabeled sample by spotting 5 ml of pre-cleared sample onto two GF/C Whatman filters; allow 1 filter to air dry (total cpm in the sample) and place the second filter in a small beaker of ice cold 10% TCA for 5 minutes
B. Transfer the filter from the TCA into a beaker containing 95% ethanol for 5 minutes; remove the filter and air dry
C. Place filters into 3 ml of scintillation fluid and count the sample
D. Determine the percent precipitable counts by dividing the cpm obtained after TCA precipitation by the total cpm in the non-TCA precipitated sample; this value should be >90%

④For unlabeled samples determine the total protein in the sample.

⑤To a microcentrifuge tube add cell lysate (20-30 x 106 TCA precipitable cpm for radiolabeled samples or 10 mg of protein for unlabeled samples), 1 mg of purified primary antibody (polyclonal or monoclonal), 15 ml of packed agarose beads of choice (i.e. Protein A-agarose, Protein G-agarose, etc.); cap the tubes, place them in a bag, and incubate at 4oC on a rocker platform for 2-24 hours; NOTE: optimal conditions for immunoprecipitation must be determined empirically for a given antibody and sample.

⑥Collect the immunoprecipiate by centrifugation in a microcentrifuge at 2500 rpm for 15 minutes, 4℃.

⑦Carefully aspirate and appropriay discard the radioactive supernatant (for radiolabeled samples); wash the pellet with 4 changes of PBSTDS, 1 ml per wash, repeating the centrifugation step with each wash.

⑧Following the final wash aspirate and discard the supernatant and resuspend the pellet in 40-50 ml of SDS-PAGE sample loading buffer.

⑨Analyze samples by SDS-PAGE

A. Visualize radiolabeled samples by exposure to X-ray film or using a phosphoimager
B. Visualize unlabeled samples by western blotting

注意事項：

⒈ This procedure can be used for cells labeled with other radioactive amino acids (e.g. 14C or 3H) or with 32P-orthophosphate; cell labeling must be carried out in a medium lacking the correct amino acid or in phosphate-free medium, as appropriate.
⒉ For western blotting use an antibody from a different species than the one used to immunoprecipitate in order to avoid detecting the precipitating antibody with the secondary antibody used for detection; alternatively, use a primary antibody that is directly conjugated to a detector system or to biotin (to be used with a streptavidin detector system).
⒊ It is useful to remove approximay 5 mm from the end of the disposable pipette tips before pipetting concentrated agarose solutions.