The following buffers and methods provide a general starting point for use with the majority of Bio-Rad reagents in Western blotting . When specific recommendations are provided on product datasheets, those instructions should always be used instead of the general guidance offered here . With specialized laboratory equipment, such as for gel electrophoresis and electroblotting, it is advisable to follow the manufacturer’s instructions .
If difficulties arise with any of our antibodies or related products, please feel free to contact antibody_tech_us@bio-rad.com as we may have additional information on file and can assist with troubleshooting.
N .B . Azide should not be added to any buffers that will be used with HrP-labeled antibodies because it will inactivate the HrP enzyme.
PBS:
TBS:
10x TBS Stock:
NP-40 Lysis Buffer:
RIPA (Radio Immuno Precipitation Assay) Buffer:
Various protease inhibitors are often added to lysis buffer to maintain the integrity of the target protein when cells are disrupted. The following table provides some recommended working concentrations.
Inhibitor | Working Concentration | Protease Inhibited |
---|---|---|
Aprotinin | 1-2 µg/ml |
Trypsin, Chymotrypsin,
Plasmin, Kallikrein
|
EDTA | 1-2 mM |
Metalloproteases (Mg2+
and Mn2+)
|
EGTA | 1 mM | Metalloproteases (Ca2+) |
Leupeptin | 0 .5-2 µg/m |
Plasmin, Trypsin, Papain,
Cathepsin B
|
Pepstatin A | 1 µg/ml | Pepsin, Cathepsin D |
PMSF (highly toxic) | 50-100 µg/ml |
Serine and Cysteine
proteases
|
Laemmli 2x Sample Buffer:
Gel Electrophoresis Running Buffer:
Transfer Buffer:
Ponceau S Stock Solution:
PBST:
TBST:
Block Ace Wash Buffer (BUF029):
Reconstitute each 4 g vial in 100 ml distilled water . Dilute the reconstituted solution 10-fold, and add Tween® 20 to a final concentration of 0 .05-0 .2% v/v.
*Use BSA or Block Ace (BUF029) to block when probing with anti-phosphoprotein antibodies, or for biotinylated primary antibodies detected with an anti-biotin secondary.
5% Nonfat Dried Milk in PBST or TBST (Blotto/BLOTTO):
3% BSA in PBST or TBST:
Block Ace (BUF029):
Chapter 4: Alternate Forms of a Protein - Prion Disease | Western Blot Protocol: Cell Lysis, Mammalian Cells |