Biotinylated Pisum Sativum Agglutinin豌豆凝集素Detection of Glycoproteins using Lectins in Histochemistry,ELISA, and Western Blot Applications
详细介绍
Biotinylated Pisum Sativum Agglutinin豌豆凝集素
isolated from Pisum sativum （garden pea） seeds Pisum sativum agglutinin is nearly identical in structure and carbohydrate specificity to Lens culinaris agglutinin. PSA has four subunits, two of approximay 17,000 daltons and two of about 6,000 daltons. Isoelectric focusing has revealed two isolectins with isoelectric points of pH 5.9 and pH 7.0. The lectin has specificity toward a-linked mannose-containing oligosaccharides, with an N-acetylchitobiose-linked a-fucose residue included in the receptor sequence. Calcium and manganese ions are required for activity. PSA has been used to fractionate cells, to isolate glycoproteins and glycopeptides, to distinguish between normal and virally transformed cells, as a T-cell mitogen, and as an inhibitor of allograft rejection.
Biotinylated Pisum Sativum Agglutinin豌豆凝集素
This biotinylated lectin conjugate is prepared from affinity-purified lectin and is optimally labeled with biotin. Essentially free of inactive lectin conjugate and containing no free biotin, this biotinylated lectin provides an ideal intermediate for examining glycoconjugates using the Biotin-Avidin System. First the biotin-labeled lectin is added, followed by the VECTASTAIN® ABC Reagent, Avidin D conjugate, or streptavidin derivative. Another possible application is in the isolation of lymphokines and other products of mitogenic stimulation. Inhibiting/Eluting Sugar: 200 mM a-methyl mannoside/200 mM a-methyl glucoside mixture
怀槐凝集素Biotinylated Maackia Amurensis LectinBiotinylated Maackia Amurensis Lectin (MAL) II Detection of Glycoproteins using Lectins in Histochemistry,ELISA, and Western Blot Applications
详细介绍
怀槐凝集素Biotinylated Maackia Amurensis Lectin
Detection of Glycoproteins using Lectins in Histochemistry, ELISA, and Western Blot Applications The following protocols offer guidelines for assay development using lectin-based detection of glycoproteins present in tissue sections, adsorbed onto microtiter plates, or transferred from electrophoretic gels onto nitrocellulose or PVDF membranes. Histochemistry: 1a. Staining procedure for paraffin sections: Deparaffinize and hydrate tissue sections through xylenes 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, fix sections 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 not use SP-2001. Block non-specific binding by incubating section with Carbo-Free™ Blocking Solution （Cat. No. SP-5040） for 30 minutes at room temperature. Blot excess blocking solution from the sections. 3. Apply biotinylated lectin at approximay 2-20 μg/ml in PBS （10 mM sodium phosphate, 150 mM NaCl, pH 7.4） to the sections and incubate for 30 minutes at 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. Apply to the sections 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 mounting in glycerol-based mounting media. ELISA: 1. Adsorb target protein to microtiter plate by placing 50-200 μl of approximay 3 μg/ml glycoprotein solution into the desired wells. Some wells may be left untreated as negative controls. Incubate at 37 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 incubate for 30 minutes at room temperature. Wash wells three times with TPBS. Vector Laboratories, Inc., 30 Ingold Road, Burlingame, CA 94010 U.S.A.
怀槐凝集素Biotinylated Maackia Amurensis Lectin
VECTOR LABORATORIES VECTOR LABORATORIES 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 the wells 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. For peroxidase, 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 cover the membrane. 3. Incubate membrane in PBS containing approximay 2-20 μg/ml biotinylated lectin for 30 minutes at 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 the membrane 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/Fluorescent Substrate for Alkaline Phosphatase （Cat. No. SK-6605） or BCIP/NBT （Cat. No. SK-5400） are recommended. Negative Controls Negative controls should be run in parallel in each of the above described methodologies to validate binding results. When applying lectins, one of the most appropriate negative controls is to preabsorb the 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 approximay 200 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 unabsorbed lectin and incubated under the same conditions. The subsequent detection procedure is followed as for the test method. In most cases the vast majority of lectin binding to the tissue section （membrane blot, etc.） will be eliminated. Some trace binding to the section （blot etc） may still be present under these conditions and probably indicates presence of secondary or tertiary sugar preferences. These negative control results should be compared with the test results to determine specificity of binding
LTL凝集素Biotinylated Lotus Tetragonolobus LectinDetection of Glycoproteins using Lectins in Histochemistry,ELISA, and Western Blot Applications
详细介绍
LTL凝集素Biotinylated Lotus Tetragonolobus Lectin
isolated from Lotus tetragonolobus seeds
Lotus tetragonolobus lectin is a family of closely related glycoproteins having 2 or 4 subunits of about 28,000 daltons. The isoelectric points differ somewhat but these isolectins probably have similar specificities toward alpha-linked L-fucose containing oligosaccharides. Although many of the binding properties of Lotus lectin are similar to those of Ulex europaeus lectin I, the binding affinities and some specificities for oligosaccharides are markedly different between these fucose-specific lectins.
LTL凝集素Biotinylated Lotus Tetragonolobus Lectin
This biotinylated lectin conjugate is prepared from affinity-purified lectin and is optimally labeled with biotin. Essentially free of inactive lectin conjugate and containing no free biotin, this biotinylated lectin provides an ideal intermediate for examining glycoconjugates using the Biotin-Avidin System. First the biotin-labeled lectin is added, followed by the VECTASTAIN® ABC Reagent, Avidin D conjugate, or streptavidin derivative. Another possible application is in the isolation of lymphokines and other products of mitogenic stimulation.
VECTOR生物素Jacalin Biotinylated JacalinDetection of Glycoproteins using Lectins in Histochemistry,ELISA, and Western Blot Applications
详细介绍
VECTOR生物素Jacalin Biotinylated Jacalin
isolated from Artocarpus integrifolia （Jackfruit） seeds Jacalin is a lectin composed of four subunits, two of approximay 10,000 daltons and two of 16,000 daltons each. This 50,000 dalton glycoprotein appears to bind only O-glycosidically linked oligosaccharides, preferring the structure galactosyl （b-1,3） N-acetylgalactosamine. This structure （the so-called “T-antigen”） is the oligosaccharide to which peanut agglutinin binds. However, unlike PNA, Jacalin will bind this structure even in a mono- or disialylated form. This lectin has been used to purify human IgA, since no other human immunoglobulin class binds Jacalin （references available upon request）. The specificity of this lectin also affords the opportunity to localize or isolate glycoproteins with O-glycosidically linked oligosaccharide side chains.
This biotinylated lectin conjugate is prepared from affinity-purified lectin and is optimally labeled with biotin. Essentially free of inactive lectin conjugate and containing no free biotin, this biotinylated lectin provides an ideal intermediate for examining glycoconjugates using the Biotin-Avidin System. First the biotin-labeled lectin is added, followed by the VECTASTAIN® ABC Reagent, Avidin D conjugate, or streptavidin derivative. Another possible application is in the isolation of lymphokines and other products of mitogenic stimulation. Inhibiting/Eluting Sugar: 800 mM galactose or 100 mM melibiose
VECTOR GNL生物素雪花莲凝集素Biotinylated Galanthus Nivalis LectinDetection of Glycoproteins using Lectins in Histochemistry,ELISA, and Western Blot Applications
The following protocols offer guidelines for assay development using lectin-based detection of glycoproteins present in tissue sections, adsorbed onto microtiter plates, or transferred from electrophoretic gels onto nitrocellulose or PVDF membranes. Histochemistry: 1a. Staining procedure for paraffin sections: Deparaffinize and hydrate tissue sections through xylenes 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, fix sections 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 not use SP-2001. Block non-specific binding by incubating section with Carbo-Free™ Blocking Solution （Cat. No. SP-5040） for 30 minutes at room temperature. Blot excess blocking solution from the sections. 3. Apply biotinylated lectin at approximay 2-20 μg/ml in PBS （10 mM sodium phosphate, 150 mM NaCl, pH 7.4） to the sections and incubate for 30 minutes at 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. Apply to the sections 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 mounting in glycerol-based mounting media. ELISA: 1. Adsorb target protein to microtiter plate by placing 50-200 μl of approximay 3 μg/ml glycoprotein solution into the desired wells. Some wells may be left untreated as negative controls. Incubate at 37 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 incubate for 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 the wells 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. For peroxidase, 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 cover the membrane. 3. Incubate membrane in PBS containing approximay 2-20 μg/ml biotinylated lectin for 30 minutes at 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 the membrane 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/Fluorescent Substrate for Alkaline Phosphatase （Cat. No. SK-6605） or BCIP/NBT （Cat. No. SK-5400） are recommended. Negative Controls Negative controls should be run in parallel in each of the above described methodologies to validate binding results. When applying lectins, one of the most appropriate negative controls is to preabsorb the 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 approximay 200 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 unabsorbed lectin and incubated under the same conditions. The subsequent detection procedure is followed as for the test method. In most cases the vast majority of lectin binding to the tissue section （membrane blot, etc.） will be eliminated. Some trace binding to the section （blot etc） may still be present under these conditions and probably indicates presence of secondary or tertiary sugar preferences. These negative control results should be compared with the test results to determine specificity of binding
The following protocols offer guidelines for assay development using lectin-based detection of glycoproteins present in tissue sections, adsorbed onto microtiter plates, or transferred from electrophoretic gels onto nitrocellulose or PVDF membranes. Histochemistry: 1a. Staining procedure for paraffin sections: Deparaffinize and hydrate tissue sections through xylenes 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, fix sections 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 not use SP-2001. Block non-specific binding by incubating section with Carbo-Free™ Blocking Solution （Cat. No. SP-5040） for 30 minutes at room temperature. Blot excess blocking solution from the sections. 3. Apply biotinylated lectin at approximay 2-20 μg/ml in PBS （10 mM sodium phosphate, 150 mM NaCl, pH 7.4） to the sections and incubate for 30 minutes at 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. Apply to the sections 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 mounting VECTOR 生物素曼陀罗凝集素Biotinylated Datura Stramonium Lectin
in glycerol-based mounting media. ELISA: 1. Adsorb target protein to microtiter plate by placing 50-200 μl of approximay 3 μg/ml glycoprotein solution into the desired wells. Some wells may be left untreated as negative controls. Incubate at 37 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 incubate for 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 the wells 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. For peroxidase, 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 cover the membrane. 3. Incubate membrane in PBS containing approximay 2-20 μg/ml biotinylated lectin for 30 minutes at 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 the membrane 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/Fluorescent Substrate for Alkaline Phosphatase （Cat. No. SK-6605） or BCIP/NBT （Cat. No. SK-5400） are recommended. Negative Controls Negative controls should be run in parallel in each of the above described methodologies to validate binding results. When applying lectins, one of the most appropriate negative controls is to preabsorb the 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 approximay 200 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 unabsorbed lectin and incubated under the same conditions. The subsequent detection procedure is followed as for the test method. In most cases the vast majority of lectin binding to the tissue section （membrane blot, etc.） will be eliminated. Some trace binding to the section （blot etc） may still be present under these conditions and probably indicates presence of secondary or tertiary sugar preferences. These negative control results should be compared with the test results to determine specificity of binding
VECTOR BPL生物素菊紫荆凝集素Biotinylated Bauhinia Purpurea LectinDetection of Glycoproteins using Lectins in Histochemistry,ELISA, and Western Blot Applications
The following protocols offer guidelines for assay development using lectin-based detection of glycoproteins present in tissue sections, adsorbed onto microtiter plates, or transferred from electrophoretic gels onto nitrocellulose or PVDF membranes. Histochemistry: 1a. Staining procedure for paraffin sections: Deparaffinize and hydrate tissue sections through xylenes 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, fix sections 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 not use SP-2001. Block non-specific binding by incubating section with Carbo-Free™ Blocking Solution （Cat. No. SP-5040） for 30 minutes at room temperature. Blot excess blocking solution from the sections. 3. Apply biotinylated lectin at approximay 2-20 μg/ml in PBS （10 mM sodium phosphate, 150 mM NaCl, pH 7.4） to the sections and incubate for 30 minutes at 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. Apply to the sections 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 mounting in glycerol-based mounting media. ELISA: 1. Adsorb target protein to microtiter plate by placing 50-200 μl of approximay 3 μg/ml glycoprotein solution into the desired wells. Some wells may be left untreated as negative controls. Incubate at 37 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 incubate for 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 the wells 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. For peroxidase, 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 cover the membrane. 3. Incubate membrane in PBS containing approximay 2-20 μg/ml biotinylated lectin for 30 minutes at 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 the membrane 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/Fluorescent Substrate for Alkaline Phosphatase （Cat. No. SK-6605） or BCIP/NBT （Cat. No. SK-5400） are recommended. Negative Controls Negative controls should be run in parallel in each of the above described methodologies to validate binding results. When applying lectins, one of the most appropriate negative controls is to preabsorb the 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 approximay 200 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 unabsorbed lectin and incubated under the same conditions. The subsequent detection procedure is followed as for the test method. In most cases the vast majority of lectin binding to the tissue section （membrane blot, etc.） will be eliminated. Some trace binding to the section （blot etc） may still be present under these conditions and probably indicates presence of secondary or tertiary sugar preferences. These negative control results should be compared with the test results to determine specificity of binding
