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How to make blocking buffer from Whatman Kit

How to make blocking buffer from Whatman Kit - Antibody Forum

How to make blocking buffer from Whatman Kit - Antibody Forum. Ask and discuss antibody suppliers, antibody related techniques and protocols, and antibody production such as using phage display libraries.


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Old 04-13-2010, 05:22 PM
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Default How to make blocking buffer from Whatman Kit



I was recently using the Whatman FAST Pak Antibody array kit to perform antibody microarrays and we have run out. The shipment is extremely delayed and I just wanted to know if anyone knows how to make a blocking buffer that is similar to the one used in this kit. I cannot get the recipe from the company, as it is imposible to attain.

Thanks,
LAura
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Old 05-14-2010, 03:57 PM
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Default Re: How to make blocking buffer from Whatman Kit

The term non-specific binding (NSB) refers to the tendency of protein molecules to adhere to a solid surface in a non-selective manner. This high background noise resulting from the non-specific binding will interfere with reporter signals to be detected from the spotted area unless the non-specific binding is blocked in an appropriate manner. The protein microarray will be immersed in a solution containing a blocking agent to block the non-specific binding sites before its contact with the intended analyte solution. A commonly used method for blocking protein non-specific binding is to treat the surface of the substrate with several animal proteins (AN) such as bovine serum albumin, casein, gelatin, etc., in buffers dissolved. Nevertheless, their use in single or in mixtures show a still discreet background noise for which, today, they are used chemical substances to modify non-spotted surface area such as polyethylene glycol (PEG), phospholipid, or polylysine to prevent non-specific binding. But a meaningful improvement has been gotten employing non animals proteins (NAP) especially from bacterial origin that has allowed to get a very low fluorescence background noise. Among these proteins, those that seem at purpose more profits, perhaps present in the blocking buffer from you used, are the E. coli lysate that now all researcher consider the "gold standard" for NSBs problems resolve.
We believe that a good protocol for blocking reagent realization to be used in microarray with thin nitrocellulose film support is below represented.
E. coli strains, better if deficient in protease (RF6333 - available "The American Type Collection ATCC #55101), in LB medium or in Terrific broth up to stationary phase has been grown. The E. Coli grows 4 times more slowly at room temperature (RT about 20 C) compared to 37 C, and culture at RT cannot carry at saturation during around 16 hrs of incubation. However, the slowest percentage of the bacterial metabolism to lower temperature can prevent formation of included bodies. After doing this first culture, transfer 1.0 ml in fresh LB medium containing 50 g/ml of ampicilin and incubate for 16 hrs between 20-37 C. Transfer 50 l of the last culture in 5 ml fresh LB medium still containing 50 g/ml of ampicilin, and incubate further 2 hrs between 20-37 C in shaking incubator growing up to mid-long phase (Abs550 nm between 0.5 -1.0). Centrifuge at 4000 g for 20 min. at 4 C and pour off the medium, and stand the centrifuge tubes in an inverted position to allow the last traces of medium to drain away. Resuspend accurately the pellet in 1/10 of the initial volume grow medium with cell lysis buffer (sodium borate 0.1 M at pH 8.0, containing 1 M NaCl - sterilize using a 0.45 m filter, and store at RT), dissolve 2 mg/ml lysozyme, and incubate for 20 min. at RT. Then dissolve 0.01 mg/ml pancreatic DNase I, and add 2 l/ml of Triton X-100 and incubate for 1 hr at 4 C, or until the turbidity clears and the viscosity decreases. For objectively evaluate degree of the lysis photometric measurement can be employed at 550 nm, at various times of the lysis process, and at the time in which Abs less 80 %, in comparison at the initial Abs of the same bacterial suspension, it is index of the lysis the most greater part of the cells. After the step, bacterial lysate were centrifuge at 8000 g for 20 min. at 4 C, carefully decant the supernatant in a fresh flask, adjust the pH to 9.0 with a 1 M NaOH, and mix accurately. Determine in the lysate the protein concentration with the BCA measurement method (see you my post “coating antibody quantitation” - Molecular Station [Only registered users see links. ] ) and adjust at desired proteins concentration with TBS Buffer (Tris-HCl 0.1 M at pH 8.0, contained 0.15 M NaCl and 0.2% [w/v] of NaN3 or other preservative), sterilize using a 0.45 m filter, and store in clear glass bottles at 4 C. Stable for several months.
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