Plant organelle/membrane isolation
Plant protein isolation
- Extraction of leaf proteins
- Diatom protein extraction
- Phenol protein extraction
- Ponceau membrane staining
- TCA acetone precipitation method
- Western blot protocol
- Western blot video tutorial
- Peptide neutralization/competition assay
- Simultaneous Western blot
- Quantitative Western blot
- Quantitative Western blot video tutorial
- Western blot troubleshooting
- Western blot using IgY
- Dot blot
- Anti-KLH antibody removal
- Yolk delipidation
Technical informationAntibody types
- Antibody purification
- Antibody purification - small amount of protein
- IgY purification methods
- Protein purification using antibodies
- Elution of antibodies from affinity columns
Protocols > ELISA
Indirect ELISA - wells are coated with the antigen (protein or peptide) and subsequently incubated with a sample containing antigen-specific antibodies (serum, yolk, affinity purified antibodies or others). This step is followed by incubation with a secondary antibody, coupled to an enzyme such as HRP or ALP, for reaction development.
Coating (first trial)
Pipet 100 µl of protein/peptide solution to each well using multi-channel pipet. Incubate over night at 4°C (cover the plate) or at 30°C for 2 hours.
No washing step needed if blocking directly follows coupling of the antigen onto a plate.
Pipet 200 µl of blocking solution to each well. Incubate at 4°C overnight or 2 hours at 30°C.
Very important step. Wash plates at least 3 times, gently.
Dilution series: from 1: 100 to 1: 100 000. Include a blank (no sample, but secondary antibody added and developing solution).
Yolk: heat to RT and dilute before applying on ELISA plate. More info here.
Incubation time: 1 h and 10 min on shaker, or 2 h with no shaking.
Incubation with secondary antibody
Follow recommendations of secondary antibody producer. Usually the higher dilution the better. At least 1: 10 000. Incubation time: 30 min on shaker, 1.5 h min with no shaking.
Many different reagents are on the market. Make sure that the reagent contains the right substrate e.g. for HRP or ALP, depending what you use. One Step substrates are working fine (no need to prepare them before the assay).
No signal or a very weak signal
High background present
Non-specific color development on the plate
Poor standard curve obtained
Recommended literature about ELISA