ELISA optimization & Techniques
ELISA Sample Protocols
- Competitive ELISA protocol
- Sandwich ELISA protocol
- Competitive ELISA protocol
- Direct ELISA protocol
- Indirect ELISA protocol
- ELISA protocol modifications
- 101 ELISA Troubleshooting tips
- Calculating & Analyzing ELISA data
- ELISA Practices & Techniques
- Immune complex assembly
- Pipetting methods & techniques
- Types of ELISA
Cell Lysis and synchronisation
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RIPA recipe protocol
RIPA (Radio Immuno Precipitation Assay) buffer is mostly used when carrying out a western blot or immunoprecipatation assay. A RIPA buffer is used in order to lyse cells and extract protein from cultured cells. RIPA buffer cell lysis enables determination of protein concentration. RIPA buffer is an ideal cell lysis reagent since it contains three non-ionic and ionic detergents.
To reduce denaturation
When you need to preserve protein-protein interactions or to reduce denaturation its recommended to use a RIPA buffer recipe without SDS (ionic detergent) or Triton X-100 (non-ionic detergent).
To prevent proteolysis & dephosphorylation
Once lysis occurs so too does protein degradation. In order to prevent/slow this process down, you must keep the samples on ice at all times and add appropriate proteinase inhibitors, added freshly each time to the RIPA lysis buffer.
Components to necessary to carry to carry out RIPA cell lysis
- Cells – Adherent or Suspension
- RIPA Buffer – Ice Cold
- Ice Cold PBS
- Freshly added proteinase Inhibitors
RIPA Buffer Recipes
1% v/v NP-40
20mM Tris-HCL pH 7.4
5mM Sodium Pyrophosphate
Freshly added proteinase Inhibitors (Leupeptin, PMSF, Sodium Ortovanadate)
50mM Tris HCL pH 7.4
50 mM NaCl
Freshly added proteinase Inhibitors (Apoprotein, Leupeptin, DTT and PMSF)
Preparation of cell lysate using RIPA Buffer
- Wash cells with ice cold PBS. Remove your cell media by spinning cells in a microcentrifuge for 5 min at 1,500 x G. Remove media by aspirating. Resuspend cell in ice cold PBS and microcentrifuge cells for 5 min at 1,500 X G.
- Aspirate PBS.
- Add ice cold RIPA Buffer (~1ml per 107 cells).
- Scrape adherent cells off the plate using your sterile pipette tip.
- The centrifugation force and time can vary depending on cell type.
- Remove from centrifuge and store on ice.
- Aspirate the supernatant into a new tube and keep on ice, discard the pellet.
- Determine protein concentration using a Bradford assay, a Lowry assay or a bicinchoninic acid (BCA). BSA can be used a standard.
- Thus once the protein concentration has been determined the samples can be frozen at -20 °C / -80 °C or prepared for loading.
Preparing samples for gel loading
- Normalise the samples to the appropriate concentration.
- Denature the protein to allow antibody detection using Lameli buffer.
- Heat block for 5 minutes 95°C.
- Load samples onto acrylamide gel
Lameli Buffer contains beta-2-mercaptoethanol which acts to reduce disulphide bonds and in turn denatures the protein. Futhermore the Lameli buffer also has a SDS component which provides the negative charge necessary for gel electrophoresis and glycerol to make the sample more dense.