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Lysate Preparation: Why is RIPA Buffer Best for Western Blot?

Why is RIPA Buffer Best for Western Blot?

In   1979,  Jaime Renart  et al. published an article entitled  “Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure,” the prelude   to  the modern Western blot (WB)    technique.

Soon after, Harry Towbin et al. went one step further and published “Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.” With that, the WB technique was officially born.

Today, WB experiments are a cornerstone of biological research. Unfortunately, it is frequently a challenge to obtain good results.

A wise man once said:

A successful WB relies upon:

10% Reagents

10% Execution

10% Luck

70% protein extraction.

Speaking of  WB,   as an original manufacturer of all our products,  Proteintech’s   R&D staff test on average over 70 samples for each product in WB. Proteintech’s   senior R&D staff will share with you our years of    experience with WB to ensure every WB is a successful one.

What Goes into a Lysis Solution?

A lysis solution contains the following  components:

1.  The   buffer system

The pH of the solution is critical. Proteins may precipitate or become unstable when the pH is outside of the physiological range. To avoid this situation, a buffer system such as Tris-HCl is recommended. Besides buffering solutions in this range, a Tris-HCl buffer preserves the physiological ionic strength and prevents the formation of insoluble products with other ions. A HEPES buffering system is another option. We recommend avoiding buffers with high concentrations of potassium, because these can precipitate proteins when sodium dodecyl sulfate (SDS) is present.

2.  Salt ions

When the salt ion concentration is too high, some proteins may precipitate. Additionally, when ion concentration is too high, a “smiley face” of band migration may result.

3. Chaotropic agents

Chaotropic agents weaken the hydrophobicity of the proteins to solubilize them. There are two kinds of chaotropic agents in a lysis buffer:

a. Urea/thiourea.  These molecules unravel hydrophobic regions by disrupting hydrogen bonding between amino acids. Usually when doing protein extraction for a WB, 6­–8M urea and/or 2M thiourea can be used.

b. Detergents. These are a broad class of surfactants. The key to their solubilizing power is their amphiphilic structure. The hydrophobic end binds to the hydrophobic portions of proteins while the hydrophilic end interacts with water, resulting in solubilization.

Ionic detergents can be further divided into cationic, anionic, and amphoteric detergents.Common ionic surfactants are SDS, DOC (sodium deoxycholate), and SLS (sodium lauryl sarcosine). Common amphoteric surfactants are CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate), and CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate). Common non-ionic surfactants are Triton X-100, Triton X-114, Tween-20, and NP40.

Note: It is critical in a WB that the number of negatively charged SDS molecules that bind to a protein is proportional to the protein’s mass, so that the migration rate is influenced only by mass. Adding cationic surfactants in the lysis buffer would disrupt the SDS-protein interaction and make the proteins migrate in the opposite direction.

Due to the complexity of protein biochemistry, it is challenging to predict the optimal surfactant to extract a given protein. Thus, experimenting with different surfactants is recommended if you encounter issues. This is especially recommended for membrane proteins.

Related: Prestained Protein Ladders
Protein Ladder (Free sample available) Broad Range Protein Ladder
Catalog no.: PL00001 Catalog no.: PL00002
Molecular weight coverage: 10-180 kDa Molecular weight coverage: 3-245 kDa
No. of markers: 10 prestained proteins No. of markers: 13 prestained proteins
Proteintech's new prestained protein marker (PL00001) is a three-color protein ladder with 10 markers covering a wide range of molecular weights from 10 - 180 kDa Proteintech's new broad range protein marker (PL00002) is a three-color protein standard with 13 markers covering a wide range of molecular weights from 3 - 245 kDa
Resolution of the prestained protein ladder in a 10-20% Tris-glycine gel (SDS-PAGE).  Resolution of the prestained protein ladder in a 4-12% Bis-Tris gel (SDS-PAGE)
4. Protease inhibitors

Tissues and cells often contain large amounts of proteases. During lysis, these are released and, in turn, can digest the target protein. Therefore, protease inhibitors are critical for preserving the target protein. Common protease inhibitors are PMSF (phenylmethylsulfonyl fluoride), Aprotinin, Leupeptin, Pepstatin, and AEBSF-HCL (4-benzenesulfonyl fluoride hydrochloride). PMSF is highly effective and is the most popular choice for lysate preparation.

Many protease inhibitors require a divalent metal ion to function, so a sequestering agent is also often used to inhibit protease activity, such as   EDTA.   In    addition, for phosphorylated target proteins, a phosphatase inhibitor such as sodium fluoride or sodium orthovanadate is needed to preserve the phosphorylated form of the protein. Sodium orthovanadate, in particular, is very effective, but needs to be activated by adjusting the pH of the solution to 10 and then boiling it until the solution is colorless. Other phosphatase inhibitors include sodium pyrophosphate and β-glycerol phosphate.

5. Reductant

Many proteins exist in multimers through disulfide bonds. Reductants disrupt these bonds so that the extracted proteins are present in monomeric form. Common reducing agents are DTT (dithiothreitol) and BME (beta-mercaptoethanol). Keeping all of this in mind, RIPA buffer is the best choice for sample lysate preparation. We have validated over 13,000 antibodies in WB, and time and time again, experience the best results using RIPA buffer. Over the years we have refined the buffer and below you will find Proteintech’s optimized version:

RIPA buffer For 1000ml
 50mM Tris HCl, PH 7.4  50ml
 150mM NaCl  8.76ml
 1% Triton X-100 or NP-40  10ml
 0.5% Sodium deoxylcholate  5g
 0.1% SDS  1g
 1mM EDTA (0.5M stock)  2ml
 10mM Naf  0.42g
 Add ddH2O to 1000ml
 Add PMSF to a final concentration of 1mM and any other protease inhibitors immediately before use. 
4 x SDS sample buffer For 1000ml
 12% SDS 120g
 25% Glycerol 250ml
 150 mM Tris.HCl (pH 7.0 1M stock) 150ml
 0.03% Bromophenol Blue 300mg
 20% β-mercaptoethanol 200ml
Add ddH2O to 50ml, aliquot and store at -20°C
20% β-mercaptoethanol, (or 500 mM DTT replaced), should be added freshly before use.

If you are experiencing issues extracting the conventional lysate protein, we recommend reading the literature, rather than randomly trying various extraction kits.


Related: Loading control antibodies
GAPDH Antibody
Catalog no.: 60004-1-Ig

GAPDH is commonly used as a protein loading control in western blot due to its consistently high expression in most cell types. This enzyme participates in several cellular events such as glycolysis, DNA repair, and apoptosis.

Proteintech monoclonal GAPDH antibodies are raised against a whole-protein antigen of human origin and have over 2,670 citations.

mouse monoclonal GAPDH antibody WB analysis of HeLa cells
Beta Actin Antibody (KD/KO validated)
Catalog no.: 66009-1-Ig 

Beta-actin is usually used as a loading control due to its broad and consistent expression across all eukaryotic cell types and the fact that expression levels of this protein are not affected by most experimental treatments.

66009-1-Ig has been cited over 1,135 publications and has wide species reactivity.

WB analysis of Jurkat cells using using beta actin antibody (66009-1-Ig)

Proteintech control antibodies are just $99 each (150 in Europe) for a 150ul size vial.

 


 

 HumanKine

 

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Posted:
31 October, 2016

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