Buffers blocking buffer

The antibody solution (1.6x10 M) and substrate solutions with various concentration from 10 M to 10 M were mixed on a BSA-coated plate. The mixed solution of antibodies and substrates was allowed to stand for 1 day at room temperature, and then transported to the ELISA plates pre-coated with BSA-hapten and BSA blocking buffer. Absorbance at 405 nm for the resulting enzymatic hydrolysis product (p-nitrophenolate) by alkalinephosphatase of the second antibody was recorded on an Immuno-Mini NJ-2300 to determine the amount of antibody bound to BSA-hapten. 

Primary antibody incubation. Prepare primary antibody cocktail in the blocking buffer (5% normal horse serum, buffer + azide), and incubate 1 h at room temperature. We typically use a mix of mouse, rabbit, goat, and/or chicken primary antibodies together see Table 5.2 for appropriate dilutions of the recommended antibodies. At this point (or during secondary antibody incubation), it is usually safe to incubate the samples overnight at 4°. 

Incubate with mouse antibodies (in the case of Inada et al. (22) it was mous e monoclonal antibodies raised against human single- and double-stranded DNA) at a dilution of 1 10 in blocking buffer for 1 h at 37°C. 

Incubate with goat anti-mouse IgM conjugated with 15-nm colloidal gold at a dilution of 1 160 in blocking buffer for 2 h at 37° C. 

Wash cultured cells attached to 35-mm plastic tissue-culture dishes in Dulbecco s PBS, then incubate in a blocking buffer consisting of BSA-PBS for 5 min, and cool to 4°C (see protocol flow chart in Fig. 1). Cooling prevents subsequent endocytosis of any added antibody reagents, as well as minimizing lateral mobility of bound antibody in the plane of the plasma membrane (see Notes 5 and 6). 

Blocking buffer IX Tris-buffered detergent (TBD 50 mM Tris, pH 7.5,200 mM NaCl, and 0.3% Tween-20) with 3% bovine serum albumin. 

Streptavidin (Boehringer Mannheim, Mannheim, Germany) blocking buffer (1 pg/mL). Biotin-conjugated alkaline phosphatase (Boehringer Mannheim), in blocking buffer, 5 pg/mL. 

Add three drops of blocking buffer to the slides and incubate at room temperature for 20 min. 

Study Print buffer Additives Protein concentration Substrate Rinse Blocking buffer... 

Once the transfer is completed, the blotting system is carefully disassembled. The filter paper and gel are removed and the membrane is placed in TBS buffer for 10 min. Finally, the membrane is soaked in blocking buffer for 60 min. 

The blocking buffer is discarded and the membrane is then incubated with primary antibody in blocking buffer for 1 h at room temperature. 

The membrane is incubated with secondary antibody in blocking buffer for 60 min at room temperature. The secondary antibody is freshly prepared for each experiment as a 1 10,000-fold dilution in blocking buffer. 

Blocking buffer (TM). TBS containing 2% dried milk powder (Marvel). 

Blocking buffer. 5% (w/v) freeze-dried nonfat milk (e.g., Marvel, Premier Beverages, Stafford, UK) in TST. Make up fresh as required. The quality of the dried milk is important, since some brands contain significant amounts of fat (see Note 4). 

Place the blot in blocking buffer (> 1 mL/cm2 membrane), and incubate on a rocking table for 1 h at room temperature or overnight at4°C, whichever is more convenient... 

Dilute the primaiy antibody in blocking buffer to give a final concentration between... 

Dilute the enzyme-conjugated secondary antibody in blocking buffer. Follow any recommendations that may be provided by the supplier regarding antibody dilution (see Note 13). 

Blocking buffer normal serum of the species in which the secondary Ab was raised, is used diluted to 5% in TBS-BSA to block nonspecific binding sites... 

To dilute the primary Ab, blocking buffer is used that contains only 1% serum... 

Then block nonspecific binding sites by floating for 20 min on blocking buffer. 

Follow this by incubation on the primary Ab (e.g., hybridoma culture supernatant, diluted appropriately in blocking buffer containing 1% serum) for 1 h... 

Blocking buffer. 5% skimmed milk powder (e.g, Marvel) in wash buffer... 

Blocking buffer (see recipe) appropriate for membrane and detection protocol... 

Place membrane in heat-sealable plastic bag with 5 ml blocking buffer and seal bag. Incubate 30 min to 1 hr at room temperature with agitation on an orbital shaker or rocking platform. 

Dilute secondary antibody HRP- or AP-anti-Ig conjugate in blocking buffer. 

Equilibrate membrane in appropriate blocking buffer in heat-sealed plastic bag with constant agitation using an orbital shaker or rocking platform. For nitrocellulose and PVDF, incubate 30 to 60 min at room temperature. For nylon, incubate >2 hr at 37°C. 

Open bag, remove blocking buffer, and add enough primary antibody solution to cover membrane. Reseal bag and incubate 30 min at room temperature with gentle rocking. 

TTBS can be stored up to 1 week at 4°C. Prepare blocking buffer containing nonfat dry milk immediately prior to use, as the milk blocking solution is not stable. 

The culture is incubated in methanol containing 3% H202 for 10 min to quench endogenous peroxidase activity. The cells are rehydrated in 70% ethanol for 2 min, followed by rinsing for 5 min in PBS. Nonspecific binding is blocked by incubating the cells for 1 hr in the blocking buffer (1% BSA in PBS). 

Add 200 pL of blocking buffer to each well. Seal plate and incubate for at least 2 h at room temperature. 

Discard blocking buffer. Wash plate three times see step 3). 

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