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 firstname.lastname@example.org 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.
10x TBS Stock:
NP-40 Lysis Buffer
RIPA (Radio Immuno Precipitation Assay) Buffer
Various proteases 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|
|EGTA||1 mM||Metalloproteases (Ca2+)|
|Leupeptin||0 .5-2 µg/m||
Plasmin, Trypsin, Papain,
|Pepstatin A||1 µg/ml||Pepsin, Cathepsin D|
|PMSF (highly toxic)||50-100 µg/ml||
Serine and Cysteine
Laemmli 2x Sample Buffer
Gel Electrophoresis Running Buffer
Ponceau S Stock Solution
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|