RIPA Buffer Recipe

What is RIPA Buffer?

RIPA (Radio Immuno Precipitation Assay) buffer is primarily used when conducting 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.

Key Takeaways

RIPA buffer is essential for lysing cells and extracting proteins, crucial for determining protein concentration.
It contains detergents to preserve protein-protein interactions, inhibit proteolysis, and prevent denaturation.
Various RIPA buffer recipes are available, tailored to specific research needs.
The process involves washing cells, adding RIPA buffer, centrifuging, determining protein concentration, and preparing samples for gel loading using Lameli buffer.

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).

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.

Materials Required for RIPA Buffer Cell Lysis

Steps	Materials
1.	Cells – Adherent or Suspension
2.	RIPA Buffer – Ice Cold
3.	Ice Cold PBS
4.	ICE
5.	Freshly added proteinase Inhibitors (Apoprotein, Leupeptin, DTT and PMSF)

RIPA Buffer Recipes

Steps	Recipe A
1.	1% v/v NP-40
2.	20mM Tris-HCL pH 7.4
3.	5mM Sodium Pyrophosphate
4.	5mM EDTA
5.	Freshly added proteinase Inhibitors (Leupeptin, PMSF, Sodium Ortovanadate)

Steps	Recipe B
1.	50mM Tris HCL pH 7.4
2.	50 mM NaCl
3.	2mM EDTA
4.	0.1% SDS
5.	Freshly added proteinase Inhibitors (Apoprotein, Leupeptin, DTT and PMSF)

Related Resources

Cell Culture Guide

Subcellular Fractionation

Sonication Protocol

Preparation of Cell Lysate Using RIPA Buffer

Steps	Procedure
1.	Wash cells with ice cold PBS
2.	Aspirate PBS
3.	Add ice cold RIPA Buffer (~1ml per 107 cells)
4.	Scrape adherent cells off the plate using your sterile pipette tip
6.	The centrifugation force and time can vary depending on cell type.
5.	Remove from centrifuge and store on ice.
7.	Aspirate the supernatant into a new tube and keep on ice, discard the pellet.
8.	Determine protein concentration using a Bradford assay, a Lowry assay or a bicinchoninic acid (BCA). BSA can be used a standard
9.	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

Steps	Procedure
1.	Normalise the samples to the appropriate concentration
2.	Denature the protein to allow antibody detection using Lameli buffer.
3.	Heat block for 5 minutes 95°C.
4.	Load samples onto acrylamide gel

Lameli Buffer

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.

Ingredient	Amount
Tris (1.0 M, pH 6.8)	10 mL
SDS	4.0 g
Glycerol	20 mL
β-Mercaptoethanol	10 mL
Bromophenol blue	0.1 g
ddH2O	Make up to 50mL

Written by Sean Mac Fhearraigh

Seán Mac Fhearraigh PhD is a co-founder of Assay Genie. Seán carried out his undergraduate degree in Genetics at Trinity College Dublin, followed by a PhD at University College Dublin. He carried out a post-doc at the Department of Genetics, University of Cambridge. Seán is now Chief Technical Officer at Assay Genie.