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=Antibody-Oligo Linking= [[Daniel Jacobsen|Back to Main]] This page describes the protocol for antibody-oligo binding used in Combo-lock. The protocol is the first step in creating antibody probes. The antibodies are generally polyclonal antibodies from R&D systems. The protocol uses Sulfo-SMCC, which is a chemical that puts a reactive group on the terminal amine of the antibody. The oligos used have a thiol group that reacts with the Sulfo-SMCC activated antibody and links the oligonucleotide to the antibody. ===Buffers=== <ol> <li>Prep 100 mL of sodium phosphate buffer</li> <ol type="A"> <li>Make 100 mL of NaH2PO4 (monobasic) by adding 13.8g to nfH2O</li> <li>Make 100 mL of Na2H2PO4 (dibasic) by adding 14.2g to nfH2O</li> <li>Mix 28 mL of monobasic and 72 mL of dibasic to make 1 M (buffer pH 7.2)</li> <li>Directions from [http://cshprotocols.cshlp.org/content/2006/1/pdb.rec8303.full?text_only=true Cold Spring Harbor] </ol></ol> ==Protocol== The protocol is taken (and slightly modified) from the [[Media:Assarsson_Homogenous_PEA_Immunoassay_PLOS_One_2014.PDF|Assarsson PEA Paper]] Additional paper provides similar but more detailed protocol [[Media:Weibrecht_Visualising_Protein-DNA_Interactions_in_situ_2012_New_Biotech.pdf|Weibrecht/Landegren 2012]] <ol> <li>Antibody Activation</li> <ol type="A"> <li>Resuspend antibodies at 1 mg/mL in PBS</li> <li>Equilibrate a 40 kDa spin column (or plate) 4 times with 100 mM phosphate buffer, pH 7.3; 250 uL, spin at 1000xg for 2 min</li> <li>Add 20 uL antibody to the column and spin for 3 min at 1000xg; collect in a new plate</li> <li>Dissolve sulfo-SMCC to 3.33 mM in 100mM phosphate buffer</li> <li>Add 2 uL sulfo-SMCC to sample and incubate for 2 hr at 4C with 3 times intermittent mixing</li> <li>Equilibrate a new 40 kDa column with 100 mM phosphate buffer with 20 mM EDTA</li> <li>Transfer antibodies to new column and spin for 3 min at 1000xg</li></ol> <li>Oligonucleotide Activation</li> <ol type="A"> <li>Resuspend oligonucleotide in 1 mM in 100 mM phosphate buffer with 20 mM EDTA</li> <li>Add 1.3 uL oligonucleotide to tube (or plate)</li> <li>Add 2.2 uL 40 mM DTT; incubate at 95C for 2 min followed by 1 hr at 37C</li> <li>Add 20 uL PBS with 20 mM EDTA <li>Remove excess DTT using two consecutive [[Media:Zeba_Spin_Desalt_Colomn_7K_MWCO_UG_Manual.pdf|Zeba 7 kDa]] columns equilibrated with 100 mM PBS</li></ol> <li>Ab-Oligo Mixing</li> <ol type="A"> <li>Mix SMCC-treated antibodies with DTT-treated oligonucleotides at 10X molar excess of oligo to Ab</li> <li>Transfer to pre-wet [[Media:SlideALyzer_MINI_Dialy_Unit_UG_Manual.pdf|Slide-A-Lyzer Mini 7 MWCO dialysis cup]]</li> <li>Dialyze in 1 L PBS with 5 mM EDTA at 4C for 2 days with one buffer exchange to PBS</li> <li>Dilute probes to 75 ug/mL in PBS buffer with 4 mM EDTA, 35 ug/mL ssDNA, 0.1% fish gelatin, and 20 mM Tris HCl</li> <li>Store at 4C</li> </ol></ol>
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