Immunoprecipitation protocol (using Precipitor™) A
Contents
1. St Neihu District Taipei City 114 Taiwan Page 1 4 Revision 1 3 Date 2013 1 9 Abnova 4 Reaction ol N 9 10 11 1 Reaction gt nN oo N PIP 2 v 2J 2 5 s 5 _ _ Reaction Reaction DIP v v 2J 2 5 s 5 _ a j Reaction Reaction j Reaction DIP 2 2 2J 2 5 5 5 _ _ Reaction v 2 S 5 na Reaction ITO Tn MOOV D AOOO Figure 2 A 96 deep well plate and its reaction areas B Reagents amp instruments Protein A Magnetic Bead Cat U0007 Antibody that specific to particular antigen Sample s that contain this particular antigen cell tissue lysate cell culture supernatant and etc 20x PBS stock 1 L NaCl 151 89 NaOH 7 09 NaHPO 27 69 Weight the salts above to dissolve in 800 ml ddH O adjust pH to 7 0 with NaOH and fill up to 1000 ml with ddH O 0 2 PBST PBS with 0 2 v v Tween 20 Blocking Buffer Weight 50 g skimmed milk power to 800 ml 0 2 PBST and stir until completely dissolve and fill up the volume to 1000 ml by adding extra 0 2 PBST 1x Elution Buffer 0 2 M Glycine 0 15 M NaCl pH 2 5 5x SDS sample buffer Add 10 g SDS 0 05 g bromophenol blue 10 4 ml 14 3 M mercaptoethanol in 25 ml 1 M Tel 886 2 87511888 Fax 886 2 87511166 9th FI No 108 Jhouzih St Neihu District Taipei City 114 Taiwan Page 2 4 Revision 1 3 Date 2013 1 9 Abnova Tris HCl pH 6 8 adj2. recommended starting value for X is 30 ul 40 ul and Y is 10 ul 2 Add Z ul 0 2 PBST to A1 Z 500 ul X Y 3 Add 500 ul blocking buffer to well A2 4 Add A ul sample containing the target antigen to A3 and then add B ul 0 2 PBST B 500 ul Aul For over expression lysate with total protein concentration of 1 ug ul the recommended starting value for A is 200 ul Add 500 ul 0 2 PBST to A4 and A5 for washing purpose Add 100 ul 1x sample buffer to A6 Tel 886 2 87511888 Fax 886 2 87511166 9th FI No 108 Jhouzih St Neihu District Taipei City 114 Taiwan Page 3 4 Revision 1 3 Date 2013 1 9 Abnova 7 Insert the 96 deep well plate into the 96 deep well plate rack please refer to figure 1 Insert 8 channel cover Strips into the cover strip rack please refer to figure 1 Execute the preset program saved in Precipitor the detail setting of the reaction steps is list in table 1 10 After the machine complete all steps place the plate on a magnetic separator and collected the supernatant contain the precipitated protein in A6 11 Boil the solution at 100 C for 5 min for subsequent Western Blot analysis Note If the precipitated protein will be used in other assay instead of western blot then replace the 1x SDS sample buffer use in the step 6 with Elution buffer and the supernatant collected in step 10 should be neutralized by adding Neutralization Buffer 1 M Tris pH 9 0 use 2 5 ul of Neutra
3. Abnova Immunoprecipitation protocol using Precipitor A Overview Immunoprecipitation IP is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that antigen This process can be used to isolate and concentrate a particular protein from a proteins mixture for example cell lysate In this guide we demonstrate a procedure of using Precipitor to immunoprecipitate the desire protein antigen from a sample by antibody coupled protein A magnetic bead Figure 1 demonstrates step by step of the precipitation reaction in Precipitor d Magnetic protein A bead C Antibody Target Non specific 1 Step 1 Step 2 Stop 3 A Mix antibody with Blocking to remove React wilh sample F oe proieln 4 beads non specific binding lysate 5 o E i i edd j E as as Step 4 Step 5 Wash with PRSIT Flute CA Mlapnetic rods Ry Cover strip rack Ct SA deep weal plate rack mE 0 channel cover strip E 66 deep well plata i oa Figure 1 The principle of the precipitation reaction in Precipitor Precipitor process one 96 deep well plate each time and each reaction utilize 6 wells hence the maximum reaction can be processed by Precipitor is 16 immunoprecipitation reactions each individual reaction area in 96 deep well plate is marked with an black open square in figure 2 7 Tel 886 2 87511888 Fax 886 2 87511166 9th FI No 108 Jhouzih
4. lization buffer for every 50 ul of elution buffer Table 1 The immunoprecipitation program for the demonstration protocol above Step Well Mixing M Collect S Rod Mixing Speed Volume Pause Vapor M 30 60 Medium 500 0 15 60 Medium 500 0 30 60 Medium 500 0 5 60 Medium 500 0 5 60 Medium 500 0 30 100 Medium 100 0 1 0 Medium 500 0 8 0 0 0 Medium 0 OFF 0 Note Please read the user manual of the machine carefully before setting the program Tel 886 2 87511888 Fax 886 2 87511166 9th FI No 108 Jhouzih St Neihu District Taipei City 114 Taiwan Page 4 4 Revision 1 3 Date 2013 1 9
5. ust the volume to 50 ml with ddH 0O and finally add 50 ml 100 glycerol mix well place at 4 C for short term storage or 20 C for long term storage Take appropriate amount and dilute with ddH O to 1x fold before use 96 deep well plate round bottom Cat U0014 8 channel cover Strip Cat U0015 Precipitor Please read the user manual before using 9 Assay procedure gt The protocol provide here is just a guide each user should determine the optimal condition for their own experiment gt The demonstration protocol below is a single immunoprecipitation reaction run in wells A1 to A6 and the precipitated protein is used in subsequent Western Blot assay gt Reaction volume of each well should not exceed 1 ml or the solution may spill out and contaminate the adjacent wells during machine running The reaction volume used in this protocol is 500 ul 1 Add X ul antibody and Y ul protein A magnetic beads 50 ug ul to A1 Several tests maybe need to be conducted to determine the optimal proportion of antibody protein A magnetic bead e g per 40 ug of human IgG use 100 ul of protein A magnetic bead as a starting proportion If the concentration of human IgG is 1 ug ul then per 40 ul X of human IgG required 100 ul Y protein A magnetic beads 50 ug ul For antibody with unknown concentration e g Abnova s MaxPab rabbit polyclonal antibody catalog Hxxxxxxxx Dxx x means any arabic number the
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