In standard Western blot (WB) approaches, denatured protein samples are separated according to their molecular weight with SDS-PAGE (polyacrylamide gel electrophoresis) and transferred to a membrane. The analysis of different organs, cell-types, and subcellular fractions like membranes, versus cytosol or different organelles may also provide useful information about differential protein expression levels. Fluorescent detection systems have a broad linear detection range that is a prerequisite for accurate protein quantification. However, they are less sensitive compared to enhanced chemiluminescent (ECL) detection.
Important: Some proteins have special requirements for good separation (e.g. unboiled samples or special gel systems). Please refer to the product specifc protocols and the remarks sections for western blotting on the respective data sheet.
Materials and reagents
- Membrane staining solution (optional): suitable staining solution (e.g. Ponceau S)
- TBS - skimmed milk: (20 mM Tris, 150 mM NaCl, 5% (w/v) skimmed milk powder), pH 7.4
- TBST - skimmed milk: (20 mM Tris, 150 mM NaCl, 5% (w/v) skimmed milk powder, 0.1% Tween 20), pH 7.4
- Washing solution A: (20 mM Tris, 150 mM NaCl, 0.1% Tween 20), pH 7.4
- Washing solution B: (20 mM Tris, 150 mM NaCl), pH 7.4
- Primary antibody
- Secondary reagent: Fluorophore conjugated secondary antibody compatible with your detection
device.
- Blotting membrane: nitrocellulose or special low fluorescent PVDF membrane.
Procedure for standard IgG based secondary antibodies
- Prepare samples (10-20 μg / lane for a 10 lane mini-gel) in reducing sample buffer according to the manufacturer recommendation. If purified proteins or peptides are loaded use 1ng-1μg / lane for a 10 lane mini gel.
- Incubate samples in sample buffer for 5 min at 95°C or 30 min at room temperature (see remarks).
- Separate the protein samples to be examined next to a molecular weight standard using SDS-PAGE. and transfer to blotting membrane by electro-blotting. Follow the manufacturer instructions for your SDS-PAGE and blotting device.
Important: Do not run the blue loading dye running-front out of the gel but stop gel before; then cut it off the gel before transfer. If that is not possible please use a compatible orange loading dye like "Orange Loading Dye" available from LICORbio.
Clean your blotting device with methanol to remove all remains from previous gel runs. Blue loading front dye may have accumulated in your device and will end up on the membrane leading to high background.
Use fresh transfer pads/sponges or whatman paper only.
Do not write with blue or black ink/ball pens on membranes. Use pencils if necessary! If you use pens, the ink will run and cover the whole blot during the antibody incubation steps. This will cause high background in the 700nm channel.
- Optional: Stain the membrane with membrane staining solution to check protein transfer. PonceauS is not recommended since the often contain fluorescent contaminants. If you need to stain after transfer please consider staining with “Revert Total Protein Stain” available from LICORbio
- Incubate in TBS - skimmed milk for 30 min on an orbital shaker at RT. Important: No detergent should be included in this step. Always use clean incubation boxes. Traces of Coomassie or PonceauS can cause high background.
- Incubate in fresh TBST - skimmed milk containing the primary antibody at the recommended dilution for at least 2h on an orbital shaker at RT or overnight at 4°C.
- Wash 3-4 times with washing solution A for 10 min each.
- Incubate with fresh TBST - skimmed milk containing the secondary reagent diluted according to the manufacturer instructions for at least 1 h on an orbital shaker.
- Wash 3 times with washing solution A for 10 min each.
- Replace washing solution A with washing solution B and let equilibrate for 5 min.
- Scan your membrane.
Procedure for FluoTag based secondary antibodies
NanoTag's unique FluoTags coupled to fluorescent dyes are ideal for time saving and quantitative western blotting with high precision.
Since single domain antibodies (sdAbs) are monovalent, they can be directly incubated with purified primary antibodies without forming non-functional clusters. Unpurified formats like antiserum, culture supernatants and ascites are not recommended.
- Prepare samples (10-20 μg / lane for a 10 lane mini-gel) in reducing sample buffer according to the manufacturer recommendation. If purified proteins or peptides are loaded use 1ng-1μg / lane for a 10 lane mini gel.
- Incubate samples in sample buffer for 5 min at 95°C.
- Separate the protein sample to be examined and a molecular weight standard using SDS-PAGE and transfer to blotting membrane by electro-blotting. Follow the manufacturer instructions for your SDS-PAGE and blotting device.
Important: Do not run the blue loading dye running-front out of the gel but stop gel before; then cut it off the gel before transfer. If that is not possible please use a compatible orange loading dye like "Orange Loading Dye" available from LICORbio.
Clean your blotting device with methanol to remove all remains from previous gel runs. Blue loading front dye may have accumulated in your device and will end up on the membrane leading to high background.
Use fresh transfer pads/sponges or whatman paper only.
Do not write with blue or black ink/ball pens on membranes. Use pencils if necessary! If you use pens, the ink will run and cover the whole blot during the antibody incubation steps. This will cause high background in the 700nm channel.
- Optional: Stain the membrane with membrane staining solution to check protein transfer.
PonceauS is not recommended since the often contain fluorescent contaminants. If you need to stain after transfer please consider staining with “Revert Total Protein Stain” available from LICORbio.
- Incubate in TBS - skimmed milk for 30 min on an orbital shaker at RT. Important: No detergent should be included in this step. Always use clean incubation boxes. Traces of Coomassie or PonceauS can cause high background.
- While blocking the membrane, pre-form complexes comprising primary antibodies and the secondary FluoTag reagent in a separate tube. For this, incubate 1 μg of your primary antibody with 4 μl of your FluoTag in 50 μl TBST - skimmed milk for 30 min in the dark.
- Incubate the membrane in fresh TBST - skimmed milk containing the primary antibody FluoTag complex at the dilution recommended by the supplier of the primary antibody for at least 2 h on an orbital shaker at RT or overnight at 4°C.
- Wash 3 times with washing solution A for 10 min each.
- Replace washing solution A with washing solution B and equilibrate for 5 min.
- Scan the membrane.
Note: This protocol has been validated in the SYSY Antibodies laboratories to ensure consistent and reliable staining results. However, for achieving the best specific signal with minimal background, the optimal antibody concentration, incubation temperature, and incubation duration should be optimized for each experiment.
