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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 .
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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 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|
|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|