Oct 24, 2020

Public workspaceNLP screening (dot blot)

DOI
dx.doi.org/10.17504/protocols.io.bkbfksjn
  • 1University of Groningen
  • iGEM Groningen 2020
a.stan.6
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DOI: dx.doi.org/10.17504/protocols.io.bkbfksjn
Protocol Citation: Andreea S 2020. NLP screening (dot blot). protocols.io https://dx.doi.org/10.17504/protocols.io.bkbfksjn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Other
The protocol is developed based on literature, but has not been tested yet.
Created: August 25, 2020
Last Modified: October 24, 2020
Protocol Integer ID: 41031
Abstract
In order to determine if the His-tagged NLP is expressed and secreted by the bacteria, we plan to use the dot blot procedure with anti-His tag anti-bodies. Dot blot is an immunological technique used for detecting proteins directly from the culture supernatant (without gel separation). Thus, the samples are directly spotted on the membrane, making it a high throughput procedure ideal for testing different secretion signals.
Materials
MATERIALS
ReagentTBS: 20 mM Tris-HCl 150 mM NaCl pH 7.5
ReagentTBS-T: 0.05% Tween20 in TBS
ReagentBSA/TBS-T: 0.1% BSA in TBS-T
On nitrocellulose membrane indicate the blotting region by drawing a grid by pencil.
Slowly applyAmount2 µL of sample on the nitrocellulose membrane at the center of the grid.

Use purified NLP-His and apply 5 points of increasing concentration of them on the nitrocellulose membrane in order to create a standard concentration curve.
Dry the membrane.
Block non-specific binding sites by washing the membrane in 5% BSA in TBS-T for 1h at room temperature.
Incubate with primary antibody diluted at the concentration recommended by the producer for 30 min. This step may require optimizing of the concentration.
Wash 3 times with TBS-T for Duration00:05:00

Incubate with the secondary antibody conjugated with HRP forDuration00:30:00 (concentration recommended by the producer).

Wash 3 times with TBS-T (15 min, 5 min, 5 min)
Wash with TBS
Incubate with ECL reagent for 1 min. Develop the blot.
Compare the intensity of the sample with the standard curve in order to estimate the concentration.