I would make sure you're incubating with the correct 2' ab with the correct detection method (e.g. fluorescence or chemiluminescence). Then probably troubleshoot your HRP reagent if it was the latter detection.

If you can afford, I would also recommend choosing a different 1' antibody. We typically use CST antibodies with success. If you expect your antibody to be expressed at low abundance, possibly try a polyclonal one. I would also make sure your incubation protocol is sound - especially cell lysis with protease inhibitor and blocking steps. We use this protocol by CST: https://www.cellsignal.com/learn-and-support/protocols/protocol-western?_requestid=560553.

If those don't work, try to do a qPCR (or do it while troubleshooting) to make sure the transcription of your protein of interest is present in the least. So you know it's the downstream that's causing troubles.

Hope it helps.

You need to explain more about the steps you took, so that we can determine the cause of the problem!

Camila Román

Western Blotting can be influenced by a variety of factors, and it can sometimes be challenging to pinpoint the exact issue. Here are some key areas to consider:

Antibody Species and Host Origin:

• Primary Antibody: Always make sure you're using the correct primary antibody for your target protein. Check the datasheet or product information to ensure specificity for your protein of interest. Additionally, the species from which the antibody was generated (e.g., rabbit, mouse, goat) is important to note.
• Secondary Antibody: The secondary antibody you use must be directed against the species of the primary antibody. For example, if your primary antibody is a rabbit anti-protein X, you should use an anti-rabbit secondary antibody. Also, ensure that the secondary antibody is conjugated to the appropriate enzyme (like horseradish peroxidase or alkaline phosphatase) for chemiluminescence or fluorescence detection.
• Detection: ECL Reagent: If you're using an ECL (enhanced chemiluminescence) reagent for detection, make sure it's fresh and that you're using the right volumes. Exposure Time: Sometimes, the signal might be too weak if the exposure time is too short, or it could be too strong and get saturated if it's too long. Experiment with different exposure times on the Chemidoc.
• Controls: Positive Control: Always run a positive control if available. This can help determine if the problem lies with the sample or elsewhere in the protocol.