Western Blotting with Fluorescently Labeled Antibodies
Licia Miller Product Manager
For western blotting, incubate the membrane with primary antibody diluted in 1×TBST (0.1% Tween®20) containing 5% w/v BSA or nonfat dry milk overnight at 4°C on a shaker. Please refer to the primary antibody product webpage for recommended primary antibody dilution buffer and antibody dilution.
Two-color western blotting requires primary antibodies from different species and secondary antibodies labeled with different dyes. Epitope overlap may cause interference and should be considered in two-color western blotting. If the primary antibodies require different primary antibody incubation buffers, test each primary antibody in both buffers to determine the buffer that is best suited for the dual labeling experiment.
Required solutions and reagents
- 20×Phosphate Buffered Saline (PBS)1 L: add 50 ml 20× PBS to 950 ml dH2O and mix.
- 10×Tris Buffered Saline (TBS)1 L: add 100 ml 10× TBS to 900 ml dH2O and mix.
- 1×SDS Sample Buffer: Blue Loading Pack or Red Loading Pack; prepare fresh 3× reducing loading buffer by adding 1/10 volume of 30×Dithiothreitol (DTT) to 1 volume of 3×SDS loading buffer. Dilute to 1× with dH2O.
- 10×Tris-Glycine SDS Running Buffer 1 L: add 100 ml 10×Running Buffer to 900 ml dH2O and mix.
- 10×Tris-Glycine Transfer Buffer 1 L: add 100 ml 10×Transfer Buffer to 200 ml methanol + 700 ml dH2O and mix.
-10×Tris Buffered Saline with Tween®20 (TBST-10×) 1 L: add 100 ml 10×TBST to 900 ml dH2O and mix.
- Blocking Buffer: 1×TBS with 5% w/v nonfat dry milk; to prepare 150 ml, add 7.5 g nonfat dry milk to 150 ml 1×TBS and mix well. Tween® 20 should not be added to the blocking buffer as it is autofluorescent and can increase nonspecific background. Tween®20 is added to subsequent diluent buffers after the blocking step.
- Wash Buffer: 1×TBST.
- Bovine serum albumin (BSA)，such as B265993.
- Primary Antibody Dilution Buffer: 1×TBST with 5% BSA or 5% nonfat dry milk as indicated on the primary antibody datasheet; to prepare 20 ml, add 1.0 g BSA or nonfat dry milk to 20 ml 1×TBST and mix well.
- Secondary Antibody Dilution Buffer: 1×TBST with 5% skim milk powder; add 1.0 g skim milk powder to 20 ml 1×TBST and mix thoroughly. (Secondary Antibodies; Anti-rabbit Ab181958 and Ab176447; Anti-mouse Ab181952 and Ab179006).
- Prestained protein standards, broad range (10-180 kDa ) , such as M665706.
- For blotting membranes and papers , 0.2 µm pore size is usually recommended.
Note: It is recommended to use reverse osmosis deionized ( RODI ) water or equivalent grade water for preparing solutions.
Experimental Steps
1. Sample preparation.
1) Aspirate the media from the culture and wash the cells with pre-chilled 1×PBS.
2) Remove PBS and add 1×SDS sample buffer (100 µl per well of a 6-well plate or 500 µl per plate for a 10 cm diameter plate) to lyse the cells. Immediately scrape the cells from the plate and transfer the extract to a microcentrifuge tube and place on ice.
3) Sonicate for 10 - 15 seconds.
4) Take 20 µl of each sample, heat at 95 - 100 ℃ for 5 minutes , and then cool on ice.
Note: The specific loading volume can be adjusted according to the protein concentration and experimental requirements. In order to avoid volume loss caused by evaporation and wall adhesion, it is necessary to prepare an appropriate amount of sample.
5) Centrifuge in a microcentrifuge for 5 minutes.
2. Sample loading and electrophoresis.
1)Assemble the pre-configured SDS-PAGE gel (10 cm×10 cm) and add electrophoresis buffer.
2) Load 20 µl onto SDS-PAGE gel (10 cm×10 cm) .
Note: The specific loading volume can be adjusted according to protein concentration and experimental requirements.
Note: It is recommended to load prestained protein molecular weight standards (M665706, 10 µl/lane) to verify protein transfer and determine molecular weight.
3) Set the voltage and electrophoresis time according to the molecular weight of the protein and start running the gel.
3.Transfer from gel to membrane
Assemble the gel and transfer device in the correct order, set the voltage and transfer time, and transfer to the nitrocellulose membrane. For detailed transfer procedures, please refer to the《General Protocol for Western Blotting》.
4. Membrane blocking and antibody incubation
Note: Capacities are for 10 cm×10 cm ( 100 cm2 ) membranes; for membranes of different sizes, adjust capacities accordingly.
1) (Optional) After transfer , wash the nitrocellulose membrane with 25 ml TBS for 5 minutes at room temperature.
2) Place the membrane in 25 ml of blocking buffer and incubate at room temperature for 1 hour.
Note : Do not add Tween® 20 to the blocking buffer.
3) Wash three times with 15 ml TBST, each time for 5 min.
4) Take 10 ml of blocking buffer and add primary antibody (according to the dilution recommended in the product data sheet) , prepare primary antibody dilution solution, place the membrane in primary antibody dilution buffer , and incubate overnight at 4°C on a shaker.
5) Wash three times with 15 ml TBST, each time for 5 min.
6) Place the membrane in 10 ml of fluorescein-conjugated secondary antibody dilution ( such as Ab181958, Ab176447, Ab181952 and Ab179006) (1 mg/ml stock solution at a dilution of 1:5000-1:25,000) and incubate at room temperature for 1 hour on a shaker.
7) Wash three times with 15 ml TBST, each time for 5 min.
5. Protein detection
1) Drain excess TBST from the membrane and allow it to dry.
Note : For fluorescent staining, it is important to ensure that the membrane is dry.
2)Please scan the membrane using an appropriate fluorescence scanner and according to the manufacturer's recommendations.
For more product details, please visit Aladdin Scientific website.