Western Blot Protocols
XenoBlot™ Reconstitution Protocol for Western Blot
Confirm your western blot setup works exactly as intended with XenoBlot™, a ready-to-use validated positive control. Follow our reconstitution protocol below to easily prepare XenoBlot™ for use in your western blot experiments.
Immunocytochemistry (ICC) allows you to locate a specific protein within or on a cell with the aid of an antibody that recognizes a particular epitope.
Indirect ICC uses a secondary antibody conjugated to a reporter. This secondary antibody binds the primary antibody. Even though this indirect method requires more steps and materials compared to the direct method, it benefits from signal amplification as multiple secondary antibodies – and their reporters –bind to the primary antibody. However, indirect ICC methods can also produce more background than direct ICC methods.
Direct ICC uses a single primary antibody to target your protein of interest. Here, the primary antibody is conjugated to a reporter such as an ATTO fluorophore. Since the direct method doesn’t require a secondary antibody, it is quicker, cheaper, and may result in less non-specific binding. However, the signal may appear weaker than the signal from indirect ICC methods, especially with proteins that have low expression levels.
- First, determine if you need to fix your cells.
- Next, chose either indirect (Option A) or direct (Option B) ICC methods.
If you have any problems, please see our extensive troubleshooting guides.
Method
- Add the ATTO-conjugated primary antibody at the appropriate dilution in ice-cold Assay Buffer.
- Optimize your WB protocol. Refer to our WB protocol for more information. Follow the product guidelines or previous experimental optimizations for a recommended dilution for your primary antibody. Determine the quantity of antibody required for two experiments.
- If your optimal antibody concentration is a 1:200 dilution, add 20 µg of antibody to a 1.5 ml Eppendorf tube containing 500 µl of PBS with 1% bovine serum albumin (BSA). Label the tube “antibody alone”.
- To a second identical tube, add 20 µg of antibody and 20 µg of blocking peptide to 500 µl of 1% BSA in PBS. Label the tube “+peptide”. Note: we recommend beginning with a 1:1 ratio between the antibody and the blocking peptide. You will need to test a series of dilutions to obtain full inhibition.
- Rotate both tubes for 1 hour at room temperature.
- Transfer the contents of each Eppendorf tube to larger tubes and add 4.5 ml of PBS with 1% BSA, 0.1% Tween-20, and 0.05% NaN3 to each tube to get a final antibody dilution of 1:200.
- Add the contents of each tube to its respective membrane test strip for parallel experiments.
- Incubate both membrane strips for 2–3 hours at room temperature or overnight at 4°C with gentle agitation.
- Proceed with the WB protocol, ensuring that you handle both the unblocked and blocked samples in the same way.
- Develop your blots and compare the signal obtained in the two test strips. The band that disappears when using the blocking peptide is specifically recognized by the antibody. Other visible bands represent non-specific antibody binding.
Jump to: Materials and reagents | Protocol steps
Why use XenoBlot™ positive control for western blot analysis?
Troubleshooting western blot experiments can be frustrating and time-consuming. A reliable positive control helps check that your reagents, antibodies, and protocol perform optimally in your experimental setup. XenoBlot™ positive control serves as a trusted benchmark, confirming that your protein of interest behaves as expected under your specific experimental conditions. By minimizing variability and troubleshooting, XenoBlot™ lets you focus on obtaining meaningful results and accurate data interpretation.
What is XenoBlot™ positive control?
XenoBlot™ is a lyophilized lysate derived from Xenopus laevis oocytes injected with RNA encoding the protein of interest. This ready-to-use lysate offers robust protein expression, serving as a validated positive control for western blot analysis. This control eliminates the need for in-house recombinant protein preparation, saving you time and effort while improving the reproducibility of your experiments.
Interpreting the western blot results with XenoBlot™ positive control
Using XenoBlot™ allows you to confirm that your antibody detects the target protein at its expected molecular weight, providing confidence in your results. A clear signal in the XenoBlot™ lane verifies that your experimental setup is working as intended.
Additional western blot controls
For a comprehensive western blot analysis, consider including other recommended controls such as blocking peptides, loading controls, and endogenous control lysates. These additional controls help ensure accurate results. Besides validated XenoBlot™ western blot controls, Alomone Labs also provides blocking peptides as negative controls.
Materials and reagents
| Item | Source |
|---|---|
| XenoBlot™ positive control | Find your XenoBlot™ positive control here |
| Deionized water (dH₂O) | - |
| 2X Laemmli Sample Buffer | - |
Protocol steps
Before starting the XenoBlot™ reconstitution protocol, make sure you have optimized your western blot protocol, including antibody selection and transfer conditions. Refer to our comprehensive western blot protocol for detailed guidance.
Start this reconstitution protocol just before loading your samples onto the SDS-PAGE gel. Use the prepared XenoBlot™ positive control as a benchmark alongside your experimental samples to confirm your experimental conditions, processing it the same way as your other samples.
- Reconstitute the lyophilized lysate by adding 50 µL of dH2O and 50 µL of 2X Laemmli sample buffer to the vial content.
⚠ Do not mix by pipetting, as this can cause foaming and inconsistent XenoBlot™ recovery.
Note that some powder may not dissolve completely at this step, but it will dissolve during heating in the next step. - Incubate the vial at 70°C for 10 minutes.
- Spin the vial for 10 seconds at room temperature before loading it on SDS-PAGE gel.
- Load 50 µL of the reconstituted XenoBlot™ control per each SDS-PAGE gel well.
Note: Each vial contains enough product for two gel lanes. - Continue with your usual western blot protocol, including electrophoresis, transfer, and immunodetection.
Note: Process the XenoBlot™ control exactly like any other sample running on your western blot.
Example Data
Expression of the α1B-adrenoceptor in GH3 cells. Cell surface detection of α1B-adrenoceptor in living GH3 cells. The cells were stained with Anti-α1B-Adrenergic Receptor (extracellular) Antibody (AAR-018) (1:100) followed by staining with goat anti-rabbit Alexa Fluor 488 secondary antibody (green). The cell nuclei were stained with the DNA dye Hoechst 33342 (blue).
Expression of the noradrenaline transporter (NET) in live intact rat pheochromocytoma (PC12) cells. Cell surface detection of NET in live intact rat PC12 cells. The cells were stained with Anti-Noradrenaline Transporter (extracellular) Antibody (AMT-002) (1:100) followed by staining with goat anti-rabbit Alexa Fluor 594 secondary antibody (red). The cell nuclei were visualized using the cell-permeable DNA dye Hoechst 33342 (blue).