Daniel:Protocols/AbHybridization: Difference between revisions

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<li>Centrifuge samples and incubate overnight at 4C</li>
<li>Centrifuge samples and incubate overnight at 4C</li>
</ol>
</ol>
==PLA Paper Protocol==
[http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0025583#s4 This paper] is actually also by Darmanis.  It covers a different style of PEA analysis.
<ol>
<li>Add probes to cell sample for final concentration of 0.5 nM</li>
<li>Incubate 1.5 hours at RT</li>
<li>Wash twice with 0.05% Tween 20 in PBS</li>
</ol>
'''PLA Buffer'''
*1 mM D-biotin  (++)
*0.1% BSA
*0.05% Tween 20
*100 nM Goat IgG (++)
*0.1 g/L salmon sperm
*5 mM EDTA
*PBS
++Note: Pretty sure these aren't necessary for the reaction I am running
'''Wash Buffer'''
*0.05% Tween 20
*PBS

Latest revision as of 00:46, 7 May 2016

Antibody Hybridization[edit]

Back to Notebook

This page details the protocol from Darmanis et al paper. The protocol is for hybridizing antibodies linked with oligonucleotides to their protein targets. The protocol will be used in validation experiments for Combo-Lock.

Darmanis Protocol[edit]

  1. Mix 1 uL cell lysate with 3 uL antibody mix
  2. Antibody Mix Contains
    1. 0.3 uL Probe A
    2. 0.3 uL Probe B
    3. 0.2 uL Incubation Stabilizer (Olink Bioscience)
    4. 2.1 uL Incubation Solution (Olink Biosicence)
  3. Centrifuge samples and incubate overnight at 4C

PLA Paper Protocol[edit]

This paper is actually also by Darmanis. It covers a different style of PEA analysis.

  1. Add probes to cell sample for final concentration of 0.5 nM
  2. Incubate 1.5 hours at RT
  3. Wash twice with 0.05% Tween 20 in PBS

PLA Buffer

  • 1 mM D-biotin (++)
  • 0.1% BSA
  • 0.05% Tween 20
  • 100 nM Goat IgG (++)
  • 0.1 g/L salmon sperm
  • 5 mM EDTA
  • PBS

++Note: Pretty sure these aren't necessary for the reaction I am running

Wash Buffer

  • 0.05% Tween 20
  • PBS