What is the protocol for O-glycosylation WB antibody detection?
Basic steps for O-glycosylation Western Blot (WB) antibody detection are as follows:
1. Sample Preparation:
Collect and prepare cell or tissue samples containing the target protein. Lyse the samples using an appropriate lysis buffer, then remove insoluble materials by centrifugation.
2. Protein Concentration Measurement:
Measure the protein concentration in the samples using a BCA protein assay kit or similar method.
3. SDS-PAGE Electrophoresis:
Select an appropriate polyacrylamide gel concentration based on the molecular weight of the proteins. Load equal amounts of protein onto the gel and perform SDS-PAGE electrophoresis.
4. Transfer:
Transfer the proteins from the gel to a polyvinylidene fluoride (PVDF) or nitrocellulose (NC) membrane using wet or semi-dry transfer methods.
5. Blocking:
Block the membrane with a 5% non-fat milk or BSA solution to prevent non-specific binding.
6. Primary Antibody Incubation:
Cover the membrane with a dilution of O-glycosylation specific antibody (usually in TBST containing 0.1% Tween 20 and 1-5% non-fat milk or BSA) and incubate overnight at 4°C.
7. Washing:
Wash the membrane with TBST to remove unbound antibodies.
8. Secondary Antibody Incubation:
Incubate the membrane with a secondary antibody labeled with HRP or another marker corresponding to the species of the primary antibody, usually for 1-2 hours at room temperature.
9. Washing Again:
Wash the membrane again with TBST.
10. Detection:
Use a chemiluminescent substrate for signal detection and perform exposure and imaging as needed.
Note: This is a general overview of the steps. Specific details such as antibody dilution ratios and incubation times should be adjusted according to the specific experimental conditions.
BiotechPack, A Biopharmaceutical Characterization and Multi-Omics Mass Spectrometry (MS) Services Provider
Related Services:
Glycosylation Site and Glycan Type Analysis
Quantitative Glycoproteomics Study
N-Glycan Modification and Site Analysis Service
O-Glycan Modification and Site Analysis Service
Post-Translational Modification Proteomics Analysis
Quantitative Glycoproteomics Study
How to order?
