Nitrocellulose membranes blocking

Tissue prints on nitrocellulose membranes (Schleicher Schuell) (0.45 pm pore size) were first stained for protein using Ponceau S (Fig. 2A,B) and photographed immediately with T-max 100 black-and-white film. The membranes were then washed in PBS to remove the stain, and incubated for 2 h with shaking in blocking solution. 

Proteins are blotted onto a nitrocellulose membrane by diffusion for 1 h at 60°C. Non-specific sites on the nitrocellulose membrane are blocked with 50 g/1 powdered milk dissolved in PBS/0.1% Tween-20. The membrane is washed in PBS/0.1%... 

In an immunoblot assay (Fig. 5-28c), proteins that have been separated by gel electrophoresis are transferred electrophoretically to a nitrocellulose membrane. The membrane is blocked (as described above for ELISA), then treated successively with primary... 

Nitro-Block enhances light output from the dioxetane substrate in reactions using AMPPD, CSPD, or Lumigen-PPD concentrate. It is required for nitrocellulose and recommended for PVDF membranes. It is not needed for Lumi-Phos 530, AP reactions on nylon membranes, or HRP-based reactions on any type of membrane. Lumi-Phos 530 is not recommended for nitrocellulose membranes. 

Add the appropriate dilution of the primary antibody to the blocked nitrocellulose membrane. Generally, 1 1000 to 1 5000 dilutions are suitable. Incubate overnight. 

Nitrocellulose membrane 0.45 i,m pore size, in sheets or rolls. Blocking solution Dissolve 3 g of low-fat dried milk in 100 mL of TBST. Make fresh. 

Store substrate at 4°C in the dark (stock solution 10 mg/ml = 23.5 mM), Do not use nitrocellulose membranes but Biodyne, Hybond N or Magnagraph nylon membranes use unpowdered gloves. Use probes at less than 20 ng/ml (Chapter 8). Signals on nitrocellulose are much weaker and require special blocking and enhancer substances (Nitro-Block Sapphire or Emerald from Tropix). Best results are on positively charged nylon also PVDF with Nitro-Block. 

B. (1982) The use ofTween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J Immunol Methods. 55, 297-307... 

The mold and punch can be constructed by a biomedical instrumentation shop, but are simple enough to be constructed in the laboratory. In theory, larger arrays can be constructed, such as for double wide (brain) slides or raw nitrocellulose membranes, measuring up to 40 by 45 mm. However, the final frozen array should not be larger than what can be cut on a cryostat or the size of the final support surface. Currently, the array block mold and punch are not available commercially. 

Following gel electrophoresis, proteins are transferred onto nitrocellulose membranes. The membranes are washed in TEST for 5 min and then blocked for 30 min at room temperature with Starting Block blocking buffer. The blots are incubated... 

CYP3A4 protein expression can be measured directly by immunodetection (LeCluyse et ah, 2000 Luo et ah, 2002). Microsomal protein (3 jig) is resolved by SDS-polyacrylamide gel electrophoresis (12% acrylamide). Resolved proteins are transferred to nitrocellulose membranes, whieh are ineubated in 3% bovine serum albumin in phosphate-buffered saline supplemented with Tween 20 (0.1 M, pH 7.4, 0.1% Tween 20) for 45 min to block nonspecific protein binding. Membranes are then treated with primary antiCYP3A4 antibody (Gentest, Woburn, MA), followed by horseradish peroxidase-conjugated antimouse seeondary antibody. The antibody-reactive CYP3A4 protein bands are visualized using enhanced chemiluminescence detection and quantitated by photodensitometry (Desai et al., 2002). 

It is wise to load the antigen with a filtration device (Schleicher and Schiill). Afterward, you fix the proteins (e.g., with ethanol/acetic acid solutions, TCA, or heating up) and block the protein binding sites of the membrane (BSA, milk powder, and so on, see Section 1.6.2). This is followed by incubations with anti-antigen and peroxidase conjugated anti-IgG antibody. You wash between the individual steps (Table 6.2). Afterward, you punch the dots on the nitrocellulose membrane into the depressions (wells) of microtiter plates. With a suitable substrate solution, a peroxidase-catalyzed color reaction develops in the wells. The color solution is transferred into new microtiter plates (to get rid of the confetti) and finally measured in the ELISA reader. A calibration curve with defined amounts of antigen quantifies the results (Becker et al. 1989). 

Spot 2-pL sample aliquots to a nitrocellulose membrane, let the membrane dry, and block with 0.5% skim milk in TBS buffer overnight. 

Tissue printing (pressure) This procedme allows the transfer of proteins to nitrocellulose membranes when a fresh cut organ or the cross sections of seeds, stems, tubers, leaves, and fruits are pressed against the membrane surface. This procedure was further modified to detect plant virus coat proteins on whole leaf blots. In this procedure, plant leaves to be blotted were placed on dry nitrocellulose membrane. The leaf and the nitrocellulose membrane were sandwiched on either side with blotting paper or one 0.75 inch thick block of plywood. The sandwich was positioned into a Carver laboratory press, and 10 000 psi of pressure was applied for 2 min. After disassembly, the membrane was air-dried for 20 min prior to standard immunodetection procedures. This procedure was shown to transfer protein uniformly. Some amount of chlorophyll was found to be transferred along with the protein, but this was not found to interfere significantly with the subsequent visualization of results. 

Treatment of nitrocellulose blots to improve detection A large number of proteins bind strongly to nitrocellulose under different experimental conditions. However, milk, Triton X-100, Nonidet P-40, and Tween-20 have all been shown to remove bound proteins. Milk, however, has been generally used to block unoccupied sites on the nitrocellulose membrane and thus prevent nonspecific binding. The sensitivity of nondenaturing blots can be increased by soaking the membrane in acidic buffer after transfer. In addition milk-stripping of antibody from membrane can be completely eliminated by exposure to acidic buffer. 

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