Greetings to all, I have attempted to detect pERK using western blotting with HEK 293T cells, but I am unable to do so. I overexpressed a gene that caused pERK to be overproduced, and I was expecting to see and detect pERK, but that did not occur. In addition to making sure the cell lysis procedure is working as best it can, I have replaced all of the buffers and meticulously completed everything else. Could someone please let me know if they have encountered this issue and how they resolved it? Many thanks in advance!
Hello,
if I understood you well, you are trying to detect the phosphorylated form of ERK protein (one of the MAKPs) but you were unable to obtain a signal using WB.
Even the control cells should have a detectable amount of pERK signal. If no signal is visible there are multiple things to improve:
When you overexpress a gene and expect to see an increase in pERK production, but are unable to detect it using western blotting, several factors could be contributing to this issue. Here are some common reasons and potential solutions:
1. **Antibody Specificity**: Ensure that the antibody used for detecting pERK is specific to the phosphorylated form of ERK. Some antibodies may cross-react with non-specific proteins, leading to false results. Consider using a different antibody with proven specificity for pERK.
2. **Overexpression Level**: It's possible that the overexpression of the gene has led to a very high level of pERK, which may saturate the detection limits of the western blot. Diluting the samples or using a more sensitive detection method, such as chemiluminescence, may help in this case.
3. **Phosphatase Activity**: Phosphorylated proteins can be rapidly dephosphorylated by phosphatases. Inhibit phosphatase activity using phosphatase inhibitors in the lysis buffer or during sample preparation.
4. **Cell Lysis and Protein Extraction**: Ensure that the cell lysis and protein extraction procedure effectively preserves the phosphorylated state of pERK. Use lysis buffers containing phosphatase inhibitors and protease inhibitors to maintain protein stability and phosphorylation status.
5. **Detection Sensitivity**: Consider optimizing the exposure time and conditions for western blot imaging to ensure that the detection system is sensitive enough to capture the signal from pERK.
6. **Secondary Antibody Issues**: Verify that the secondary antibody used for detection is functioning properly and is compatible with the primary antibody.
7. **Control Samples**: Include positive and negative control samples to validate the specificity and sensitivity of the western blotting procedure.
8. **Alternative Assays**: Consider using alternative assays such as immunofluorescence or immunoprecipitation to validate the presence of pERK.
By addressing these factors, you may be able to troubleshoot the issue and improve the detection of pERK in your experimental setup
It is not feasible to overexpress, but discussing the activation of pERK is an option. Visualization may have been hindered due to the lack of specificity of the antibody employed, particularly in the HEK 293T cell line.