IP/Co IP Operation Method

1.  Wash The Adherent Cells In The Tissue Culture Tray Or Bottle Twice With Cold PBS Buffer, And Then Drain The PBS. The Non Adherent Cells Were Also Washed With PBS, And Then Centrifuged For 5 Minutes In A Desktop Centrifuge At 800 - 1000 Rpm To Precipitate The Cells. 2.  Add Cold RIPA Modified Buffer To Cells (add 1 Ml To Every 10 Cells/100 Mm Tissue Culture Plate/150 Square Cm Tissue Culture Plate Bottle, Add 0.5 Ml To Every 5 Cells × 106 Cells/60mm Tissue Culture/75m2 Tissue Culture Bottle). 3.  Use One Of The Rubber Lake Brooms Or Plastic Cell Scrapers That Have Been Cooled In Ice Distilled Water, Scrape Off The Adherent Cells In The Tissue Culture Plate Or Bottle, And Transfer The Cell Suspension To The Centrifuge Tube. 4.  Place The Centrifuge Tube On The Incubator Of The Shaker Or The Fixed Orbit Oscillator, And Gently Shake The Suspension At 4 ℃ For 15 Minutes To Lyse The Cells. 5.  Then, Use The Pre Cooled Centrifuge To × G Centrifuge The Cell Lysate For 15 Minutes. After Completion, Immediately Transfer The Supernatant To A New Centrifuge Tube And Discard The Sediment From The Old Centrifuge Tube. 6.  When Preparing Protein A Or G Agarose (or Agarose Gel), Use PBS To Clean Agarose Beads Twice, And Maintain 50% Of The Slurry In PBS. In The Process Of Operating Agarose Beads, We Suggest To Cut The Tip Of The Straw To Operate, So As To Avoid The Fragmentation Of Beads. 7.  Add 100 μ L Of Protein A Or Protein G Agarose/agarose Bead Slurry (50%) To Each Ml Of Cell Lysate To Pre Clarify The Cell Lysate; To Do This, Place It On A Shaker Thermostat Or A Fixed Orbit Oscillator And Incubate It At 4 ℃ For 10 Minutes. The Pre Clarified Cell Lysate Can Reduce The Non-specific Binding Caused By The Interaction Between Cell Protein And Agarose/agarose Gel In Later Assay Experiments. 8.  Centrifuge 14000 X G At 4 ° C For 10 Minutes To Remove Protein A Or Protein G Beads, And Transfer The Supernatant To Another New Centrifuge Tube. 9.  To Determine The Protein Concentration Of Cell Lysates, It Can Be Determined By Bradford. However, Before Determining The Protein Concentration, The Cell Lysate Should Be Diluted At Least 1:10 Times, Because The Detergent In The Lysate Buffer Will Be Interfered By Coomassie Based Reagents. 10.  Dilute The Total Amount Of Cell Protein Of The Cell Lysate With PBS To About 1 μ G/μ L To Reduce The Concentration Of Detergent In The Buffer Solution; However, If A Cell Model With Low Protein Level Is Used, It May Be Necessary To Have More Concentrated Cell Lysate (i.e. 10 μ G/μ L) To Precipitate The Protein. At This Time, Add The Recommended Amount Of Immunoprecipitation Antibody (see Details In The Data Sheet) To 500 Ul Of Cell Lysate (i.e. 500 μ G). However, In Order To Obtain Any Target Protein For Immunoprecipitation, The Optimal Amount Of Antibody Should Be Used, Which Must Be Determined According To The Experimental Experience Of Each Cell Model. 11.  Shake The Cell Lysate Antibody Mixture Gently At Room Temperature With A Shaker Incubator Or A Fixed Orbit Oscillator For 2 Hours Or Overnight At 4 ℃. Incubation At Room Temperature For 2 Hours Is Recommended For Incubation Of Immunoprecipitated Active Phosphokinase Or Phosphohydrolase. 12.  The Protein Antibody Immune Complex Can Be Captured By Adding 100 Ul Of Protein A Or G Agarose/agarose Bead Slurry (equivalent To 50 Ul Of Tightly Pressed Beads), And Gently Shaking For 1 Hour At Room Temperature In A Shaker Incubator Or Orbital Oscillator, Or Overnight At 4 ℃. In Many Cases, If We Add 2 Ul Of Bridging Antibody (e.g. Rabbit Anti Mouse IgG), We Can Enhance The Ability To Capture This Immune Complex. This Method Is Especially Important For Some Antibodies With Poor Binding Protein A, Such As Mouse IgG1 Or Chicken Antibody. 13.  Then, Collect Agarose/agarose Beads By Pulse Centrifugation (centrifugation At 14000 Rpm For 5 Seconds). Discard The Supernatant And Rinse The Beads 3 Times With 800 Ul Cold Modified RIPA Buffer Solution. Occasionally, Washing Agarose/agarose Beads With This Modified RIPA Buffer Will Peel Off The Protein Antibody Immune Complex That Binds To Them. In This Case, It Is Recommended To Wash With Mild PBS Buffer. 14.  Finally, Suspend Agarose/agarose Beads In A Sample Buffer Of Twice The Concentration Of 60 Ul, And Mix Gently; This Suspension Will Be Sufficient For Three Electrophoresis Swimming Lanes. Boil Agarose/agarose Beads For 5 Minutes To Dissociate Their Protein Antibody Immune Complexes From The Beads. The Beads Were Collected By Centrifugation, And The Supernatant Was Detected By Polyacrylamide Gel Electrophoresis. Or Transfer The Supernatant Into A New Centrifuge Tube And Store It At - 20 ℃ For Future Use. If It Is Frozen Supernatant, It Should Be Boiled Directly For 5 Minutes Before Gel Electrophoresis& Nbsp; 2： Nodes To Be Noted: 1. If The Prepared Cell Lysate Is Used, Please Start From Step 5. 2. Most Of The Immunoprecipitation Experiments Should Be Conducted At 4 ℃, And Protease Inhibitors And Or Phosphatase Inhibitors Should Be Added To The Lysate In Advance According To The Purpose Of The Experiment 3. The Success Of The IP/Co IP Experiment Is Closely Related To The Quality Of Protein Samples (antigen Quality). Selecting An Appropriate Protein Preparation Scheme And Improving The Quality Of Protein Samples Are Necessary Conditions For The Success Of The Experiment.