Protein Purification for WB

Last updated: 1/12/12 By: Tim Starr

Overview

Basically trying to break apart cells, get rid of membrane and nucleic acids to purify a solution of proteins. For WB, need about 20 to 50 µg of total protein in a volume less than 25 µl per lane. 1e6 cultured mammalian cells should produce roughly 200 µg protein, however, I find I get much less. Different methods for disrupting cells.

• Sonication: Resuspend in PBS and sonicate
• Extrusion: Resuspend in PBS and repeatedly pass through a 21-gauge needle.
• Homogenization: Put tissue in 5 ml snap tube with 600 µl Lysis buffer and homogenize on wet ice using 5 x 30 sec bursts at 20,000 rpms.
• Emulsification: Vortex with a buffer containing ionic or non-ionic detergent and mix cells 15 minutes at 4°C.

The first two methods preserve protein-protein interactions and the "native" conformation of the protein. The third method will disrupt protein-protein interactions and disrupt the native conformation, resulting in "denatured" proteins. The method chosen should depend on the use. If doing Westerns, check which method is best for the antibody (i.e. some antibodies require denatured proteins).

Protocol 1: Protein lysate prep using RIPA buffer

• Wash adherent cells twice in the dish or flask with ice-cold PBS and drain off PBS.
• Wash non-adherent cells in ice cold PBS and centrifuge at 800 to 1000 rpm in a table-top centrifuge for 5 minutes to pellet the cells. TIP: When working with large volumes of non-adherent cells, the cells may not be cooled quickly enough to maintain the activity of the protein being studied. In this case, pour the cell suspension into a mixture of an equal mass of 2 x PBS and ice, then collect the cells by centrifugation and perform the lysis as described above.
• Add ice-cold modified RIPA buffer to cells (1 ml per 10e6 cells/100 mm dish/150 cm2 flask; 0.5 ml per 5e6 cells/60 mm dish/75 cm2 flask).
• Scrape adherent cells off the dish or flask with either a rubber policeman or a plastic cell scraper that has been cooled in ice-cold distilled water.
• Transfer cell lysate into a centrifuge tube.
• Gently rock the suspension on either a rocker or an orbital shaker in the cold room for 15 minutes to lyse cells. Can also vortex periodically.
• Note: If there is a lot of DNA, your lysate will have a big glob of gooey DNA that will not pellet when spun. To get rid of this glob of goo you need to shear the DNA either by sonication, or by repeatedly running through a 21 gauge needle. Otherwise, when you spin in the next step, there will be no pellet.
• Centrifuge the lysate at 14,000 x g in a precooled centrifuge for 15 minutes.
• Immediately transfer the supernatant to a fresh centrifuge tube and discard the pellet.
• Supernatant contains mostly cytoplasmic proteins while pellet contains nuclei & debris. Nuclear proteins are still present, although at a reduced amount.
• Dilute the cell lysate at least 1:10 before determining the protein concentration because of the interference of the detergents in the lysis buffer with the Coomassie-based reagent.
• Aliquot sample and store at -20°C for up to a month. For long-term storage freeze at -80°C.

Lysis Buffer recipe (a.k.a. RIPA buffer - radioimmunoprecipitationassay

Variations to the above include substituting Triton X for Nonident P40, adding EGTA at 1 mM. Do not use Na-deoxycholate for lysates that will be used for kinase assays because it may denature the proteins.

Protease inhibitor solution

Protease Cocktail is a pill (Roche Cat # 1 697 498) that is dissolved in 2 ml of H2O, leftover can be frozen at -20°C. These pills require the buffer to be at 1 mM EDTA. An alternative to this pill is to use a cocktail of aprotinin, leupeptin, and pepstatin by adding 1 µl of each at 10 mg/ml.

PMSF is extremely unstable (30 min) in aqueous form. So stock is made up in isopropanol and stored at –20° C and then added just before using. To make 10 ml of 100 mM add 0.174 g PMSF to 10 ml isopropanol

Na3VO4 is added to inhibit removal of phosphate groups. The activity of Na3VO4 can be substantially increased by the following activation procedure:

• Make 200 mM stock (0.368 g/ 10 ml H2O)
• Adjust to pH 10 using HCl or NaOH
• Boil until colorless (approx 10 min)
• Adjust to pH 10 again
• Repeat boiling
• Repeat the previous two steps until liquid is colorless and pH is stable
• Aliquot and store at -20° C.

Quantify protein concentration using a Bradford Assay with Coomassie blue (see quantitation protocol)

Store samples at -80°C. Boil for 10 minutes before loading onto gels.

Protocol 2: Protein lysate prep using Laemmli/SDS buffer

• Pellet cells. Generally done in eppendorfs containing the number of cells you want to load in one lane
• Resuspend pellet in 60 µl boiling 5x Laemmli/SDS buffer

Laemmli/SDS Lysis Buffer recipe 1.0 ml of 5x

• Just before using add either 30 µg Dithiothreitol (DTT) or 20 µl Beta-mercaptoethanol (BME) as a reducing agent
• Store samples at -80°C or run directly on SDS-Page gels