Sep 08, 2020
Slot/Dot Blot protocol.
- 1Rutgers University
- XPRIZE Rapid Covid Testing
Protocol Citation: beldenwj 2020. Slot/Dot Blot protocol.. protocols.io https://dx.doi.org/10.17504/protocols.io.bk3mkyk6
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: Working
We use this protocol in our lab and it is a great time saver.
Created: September 08, 2020
Last Modified: September 08, 2020
Protocol Integer ID: 41805
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Abstract
This is a simple slot/dot blot protocol for detecting antigen on a membrane. Using the method outline here, one can achieve results in under 90 min because it eliminates the need for running a gel, transfering the protein in a transfer apparatus, blocking, secondary antibody incubation and the washes between primary and secondary antibodies.
Set up Slot/Dot blot apparatus
Set up Slot/Dot blot apparatus
5m
5m
1. Assemble 3 sheets of cut GB003 blotting paper pre-wetted with transfer buffer (25 mM Tris, 200 mM Glycine, 20% methanol) to the dot blot apparatus.
2. Pre-wet a cut sheet of PVDF in 100% methanol for 30 sec and then wash the PDVF in transfer buffer for 2 min.
3. Lay the PVDF membrane on the GB003 blotting paper and seal the slot/Dot blot apparatus.
5m
Add sample
Add sample
5m
5m
1. Apply individual (or pooled) saliva or processed nasal swabs to the slot/dot blot apparatus making note of sample location.
2. Turn on the vacuum source so the sample get drawn onto the membrane.
3. Disassemble the slot/dot blot apparatus and place the membrane back in 100 % methanol.
4. Air dry the membrane for 5 minutes.
5m
Incubate with HRP-conjugated antibody, wash and develop
Incubate with HRP-conjugated antibody, wash and develop
1h
1h
1. Once the membrane is dry, add it to 10 ml of TBST (20 mM Tris, 150 mM NaCl, 0.1% Triton X-100, pH 7.5) (or PBST) containing HRP-conjuated antibody and incubate with gentle shaking 40 minutes.
2. Wash the blot 4 times for 5 minutes with 10 ml of TBST
3. Add ECL or colorimetric substrate, develop for 2-5 min, and image blot.
4. Record the results.
1h