VECTOR EEL生物素大葉黃楊Europaeus凝集素
The following protocols offer guidelines for assay development using lectin-based detection of glycoproteinspresent in tissue sections, adsorbed onto microtiter plates, or transferred from electrophoreticgels onto nitrocellulose or PVDF membranes.Histochemistry:1a. Staining procedure for paraffin sections: Deparaffinize and hydrate tissue sections throughxylenes or other clearing agents and graded alcohol series and rinse for 5 minutes in tap water.If required, retrieve antigens using the Antigen Unmasking Solution （H-3300 or H-3301）.1b. Staining procedure for frozen sections: Air dry sections. Immediay before staining, fixsections with acetone. Transfer slices to buffer. If endogenous enzyme activities are present,inactivate using appropriate methods.2. Perform Streptavidin/Biotin blocking if required following kit instructions （SP-2002）. Do notuse SP-2001. Block non-specific binding by incubating section with Carbo-Free™ BlockingSolution （Cat. No. SP-5040） for 30 minutes at room temperature. Blot excess blocking solutionfrom the sections.3. Apply biotinylated lectin at approximay 2-20 μg/ml in PBS （10 mM sodium phosphate, 150mM NaCl, pH 7.4） to the sections and incubate for 30 minutes at room temperature. Wash withTPBS （PBS + 0.05% Tween™20）.4. Prepare VECTASTAIN®® ABC （peroxidase, Cat. No. PK-6100） or VECTASTAIN® ABC-AP（alkaline phosphatase, Cat. No. AK-5000） reagents according to the kit instructions. Apply to thesections and incubate for 30 minutes at room temperature. Wash with TPBS.5. Apply an appropriate precipitating substrate for the enzyme system used in step 4. For peroxidase,ImmPACT™ DAB （Cat. No. SK-4105） is recommended; for alkaline phosphatase, Vector® Red（Cat. No. SK-5100）. Rinse in tap water.6. Counterstain （optional）, clear and mount. For galactose or GalNAc-specific lectins avoid mountingin glycerol-based mounting media.ELISA:1. Adsorb target protein to microtiter plate by placing 50-200 μl of approximay 3 μg/ml glycoproteinsolution into the desired wells. Some wells may be left untreated as negative controls. Incubate at37 oC for 1 hour. Wash wells three times with TPBS （PBS + 0.05% Tween™20）.2. Block non-specific binding by filling each well to the brim with Carbo-Free™ Blocking Solution（Cat. No. SP-5040） for 30 minutes at room temperature. Wash wells three times with TPBS.3. Apply 50-200 μl of approximay 2-20 μg/ml biotinylated lectin in PBS to the wells and incubatefor 30 minutes at room temperature. Wash wells three times with TPBS.
4. Prepare VECTASTAIN®® ABC （peroxidase, Cat. No. PK-6100） or VECTASTAIN® ABC-AP（alkaline phosphatase, Cat. No. AK-5000） reagents according to the kit instructions. Apply to thewells and incubate for 30 minutes at room temperature. Wash wells three times with TPBS.5. Apply an appropriate non-precipitating substrate for the enzyme system used in step 4. Forperoxidase, ABTS （Cat. No. SK-4500） is recommended; for alkaline phophatase, pNPP （Cat. No.SK-5900）.6. Quantify the colored reaction product by spectrophotometry.Western Blot:1. Perform electrophoresis and transfer proteins to a membrane according to standard procedures.2. Block non-specific binding by incubating the membrane in Carbo-Free™ Blocking Solution （Cat.No. SP-5040） for 30 minutes at room temperature. Use a sufficient volume to compley coverthe membrane.3. Incubate membrane in PBS containing approximay 2-20 μg/ml biotinylated lectin for 30 minutesat room temperature. Wash with TPBS （PBS +0.05% Tween™20）.4. Prepare VECTASTAIN®® ABC （peroxidase, Cat. No. PK-6100） or VECTASTAIN® ABC-AP（alkaline phosphatase, Cat. No. AK-5000） reagents according to the kit instructions. Incubate themembrane in the reagent for 30 minutes at room temperature. Wash with TPBS.5. Apply an appropriate substrate for the enzyme system used in step 4. For peroxidase, DuoLuX™Chemiluminescent/Fluorescent Substrate for Peroxidase （Cat. No. SK-6604） or ImmPACT™ DAB（Cat. No. SK-4105） are recommended; for alkaline phosphatase, Chemiluminescent/FluorescentSubstrate for Alkaline Phosphatase （Cat. No. SK-6605） or BCIP/NBT （Cat. No. SK-5400） arerecommended.VECTOR EEL生物素大葉黃楊Europaeus凝集素
Negative ControlsNegative controls should be run in parallel in each of the above described methodologies to validatebinding results. When applying lectins, one of the most appropriate negative controls is to preabsorbthe lectin with a concentration of a defined sugar, with which, the lectin has a known high affinity.Vector Labs offers a series of sugars that are intended for such a purpose.The lectin is diluted to a suitable working concentration in a solution containing approximay200 mM to 500 mM of the sugar. This mixture is left to bind at room temperature for 30 to 60 min.Following this absorption incubation, the mixture is substituted into the procedure in place of the unabsorbedlectin and incubated under the same conditions. The subsequent detection procedure is followedas for the test method. In most cases the vast majority of lectin binding to the tissue section （membraneblot, etc.） will be eliminated. Some trace binding to the section （blot etc） may still be present underthese conditions and probably indicates presence of secondary or tertiary sugar preferences. These negativecontrol results should be compared with the test results to determine specificity of binding
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