Antibody dilution buffer

Standard antibody dilution buffers such as PBS with BSA or normal goat serum as blockering agents can be used, provided generous detergent (0.5-1% triton X-100) is also included. 

Add 5 pi detection antibody to 2.5 ml lx Antibody Dilution Buffer, per membrane used. 

Antibody dilution buffer 100 mA/Tris-HCl, 150 mM NaCl, pH 7.5, containing 1% (w/v) blocking reagent (from Boehnnger Mannheim). 

Incubate the sections in antibody dilution buffer, containing 1 % blocking reagent, for 10 min... 

Antibody dilution buffer 10% horse serum (Sigma) and 0.1% (v/v) saponin in PBS. 

Prepare secondary antibodies dilutions (1 500 dilutions for all Alexa Fluor and Cyanin 5-conjugated antibodies) in antibody dilution buffer and distribute them as 50-pL drops. Prepare four beakers, two containing 50-100 mL of 0.1% saponin-PBS, one containing PBS, and one containing bi-distilled water. 

Triton X-100 permeabilization can help extract epitopes from intracellular membranes or cytoskeletal structures and yield to better staining. Because the Triton X-100 effect on cell membranes is irreversible, it is not required to keep it in the antibody dilutions buffers. 

Incubate sections for 30 60 min at room temperature or overnight at +4°C with primary antibody diluted in buffer. If your primary antibody is biotinylated, you may omit the next two steps. 

The number and duration of washing steps can help with the reduction of background. As a rule of thumb, the volume of the wash should be twice the antibody dilution volume, and standard wash time should be 5x 4 min or 3x 10 min. Washing solutions used are PBST or TEST as buffering base, but Tween-20 is sometimes omitted when there is the chance that a low affinity antibody or a weakly bound antigen could be washed away. 

Pipet 100 pi of the antibody or antiserum dilution (for titer determination a serial dilution in PBS), blank (buffer without antiserum) and, if available, controls into the respective wells and incubate on a shaker at RT for 30 min. Remove the antibody dilution and wash three times with 250 pi of Soln. B each. 

Dilute rabbit anti-AFBi serum 1 10,000 using 2 pL of antibody in 20 mL of dilution buffer. Optimal dilution of rabbit serum is determined in a preliminary experiment using a range of serum dilutions against a range of AFBi concentrations (see Notes 7 and 8)... 

Dilute rabbit anti-IgG-peroxidase conjugate 1 5000, using 4 pL of antibody in 20 mL of dilution buffer, approx 5 min before use, and leave it in ice (see Note 9). 

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). 

Resuspend the pellet in 0.5 mL of antibody incubation buffer and 25 pL of monoclonal antibody. The dilution of monoclonal antibody will vary according to the preparation (see Notes 5 and 6) The monoclonal antibody used in the author s studies is derived directly from the supernatant from the antibody producing cell line Incubate the tubes for 1 h at room temperature with occasional mixing (see Note 7)... 

It is suggested that new monoclonal antibodies be tested in several dilutions higher than those recommended by the vendor, using 0.05 M Tris buffer (pH 6.0 and 8.6) (Boenisch, 1999). The highest dilution and the pH at which maximal staining occurs should be determined for routine use of the new antibody. This approach frequently allows for the use of antibody dilutions much higher than those recommended by the supplier. 

The sections are incubated in a primary antibody (diluted appropriately) for 72 hr at 4°C in a sealed humid chamber the incubation is carried out by applying droplets of the antibody to the sections. After being rinsed in the buffer, the sections are incubated for 90min in secondary antiserum diluted 1 50 with PBX (0.3% Triton X-100, 0.01% sodium azide and 0.1 M phosphate buffer) and then treated for 1 hr under agitation in peroxidase-antiperoxidase (PAP), diluted 1 100 with PBX, in a sealed humid chamber in both cases. 

Free-floating sections (40 xm) of paraformaldehyde-fixed tissues are rinsed three times for 5 min each in 0.1 M sodium phosphate buffer (pH 7.4) (Jiao et al., 1999). They are transferred to 10-15 mM sodium citrate buffer (pH 8.5-9.0) preheated in a water bath kept in a conventional oven at 80°C for 30 min. The sections are allowed to remain in this buffer for 30 min to cool to room temperature. Following rinsing three times for 5 min each in the same buffer, the sections are treated by immersion in 0.3-3% nonfat dry milk in 0.1 % sodium azide for 30-60 min. The sections are then incubated in the primary antibody, diluted with a mixture of 0.3% Triton X-100, 0.01% sodium azide, 0.1 M sodium phosphate buffer (pH 7.4) (PBX), and 5% normal horse serum for 72 hr at 4°C under constant agitation. 

Malignant lesions and normal tissue biopsies (colon) are fixed with formalin and embedded in paraffin (Hernandez-Blazquez et al., 2000). Paraffin sections (4 p,m thick) are deparaffinized, rehydrated, and rinsed in PBS. They are placed in 0.1 mM citrate buffer (pH 3.4), heated for 10 min in a microwave oven at full power (720 W), and washed in PBS. The slides are immersed in 2N HC1 for 2 hr at 37°C and then rinsed in PBS. The sections are covered with 100 p,l ofhybridoma supernatant containing anti-5-MeCyd monoclonal antibody (5 (xg/ml) and incubated for 1 hr at room temperature with a biotinylated goat antimouse secondary antibody diluted 1 200 in PBS containing 0.1% BSA. 

Primary antibody diluted with inappropriate buffer. Use of PBS or TBS as an antibody diluent. Lack of stabilizing or carrier protein. Detergent in diluent. Check formula and compatibility of antibody diluent. A change of ion content and/or pH of the antibody diluent can cause a diminution in the sensitivity of the antibody. Addition of NaCI should be avoided. This problem is primarily seen with monoclonal antibodies. 57-60... 

The separation by ion exchange can also be done using cationic sorbents. In this case and in the presence of mild dissociating agents (dilute buffered solutions of ethylene glycol and or urea), antibodies will be found in the flowthrough and impurity traces as well as traces of protein A will be adsorbed by ion exchange. 

At this point, the anti-/3-galactosidase antibody (primary antibody) diluted in blocking buffer is added to the well. During the incubation, it will bind to the /3-galactosidase adsorbed to the bottom of the well. Several washes are then performed to remove primary antibody that may have bound nonspecifically to proteins on the bottom of the well other than /3-galactosidase. As in the Western blot experiment, the wash solution contains a small... 

Fill the well in rows A through E completely with hlocking buffer. Incubate for 30 min at room temperature. During this incubation, the BSA in the blocking buffer will bind to all of the sites on the bottom of the well not already bound with /3-galactosidase. During the 30-min incubation, prepare the antibody dilutions described in step 4. 

Using a fresh micropipette tip, follow the same procedure (steps 6 and 7) to do twofold serial dilutions of the Jl monoclonal antibody across row E. Remember that well IE will contain no primary antibody. NOTE At this point, all of the wells in rows A through E should contain 75 pd of either blocking buffer or blocking buffer plus a particular antibody dilution. 

After the membrane has been incubated with the primary antibody (diluted in blocking buffer), the membrane is washed in blocking buffer containing polyoxyethylenesorbitan monolaurate (Tween 20). This non-ionic (nondenaturing) detergent will disrupt hydrophobic interactions between the primary antibody and other proteins on the membrane (be-... 

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