VECTOR B-11335 EEL大叶黄杨Europaeus凝集素Biotinylated EEL

大叶黄杨Europaeus凝集素Biotinylated EEL
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.

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