Running a positive control cell line (where it has been established that the target is present) will help to distinguish between absence due to biology, or technical factors.

The human protein atlas can be a good starting point for identifying potential controls.

Best of luck!

Sam

Are you reliably detecting housekeeping genes like actin/tubulin etc?

You might have already tried these.

Ponceau stain to confirm if sufficient protein is being transferred.

Try using a different antibody, prefer monoclonal over polyclonal antibodies.

To further validate the antibodies, you can try dot blot using lysate and or antigen peptide (used to generate those antibodies). You can certainly try IHC which will be a quick way to answer the expression. You might need different antibodies than the exclusively made for WB. Best!

Hello Jamila Hijazi,

You could increase the sensitivity of Western Blot technique to detect proteins with low expression by adopting the following measures.

1. First and foremost, ensure that you have an efficient extraction method of your protein of interest in place. Some proteins are particularly hard to extract due to their location, for instance, proteins that are commonly located in the nucleus such as transcription factors. These proteins rely on efficient lysing of not only the cell membrane but also the nuclear envelope to get released into the lysate. RIPA lysis buffer is the recommended buffer as it contains SDS, a harsher detergent to ensure complete lysis of these intracellular compartments. Another way out to increase extraction efficiency of your protein of interest would be to use fractionation kits that may help to extract specific parts of the cell for example, mitochondria or nucleus thereby enriching your sample.

2. Make sure that you have a total transfer of your protein of interest onto your membrane before probing. If your target is of low abundance, any loss in the transfer step may mean a total loss of your signal. You need to optimize the transfer protocol. For instance, high molecular weight proteins would require longer transfer time. You may use PVDF membrane as it has a higher binding capacity in comparison to nitrocellulose. 

3. The next factor that you may consider is optimizing your antibody concentration. Do not assume that increasing the concentration of the primary and secondary antibodies will lead to a better signal, which is not always the case, since too much secondary antibody can lead to high background and signal variability.

4. Finally, use higher sensitivity detection methods. A highly effective way of increasing sensitivity is by using enhanced sensitivity substrates. These are designed to produce higher levels of chemiluminescence signal compared to the standard substrates. Using a substrate that provides a brighter signal will help increase the chance of visualizing the low abundance proteins. Please note that when you use high-sensitivity ECL, using too much secondary antibody can lead to high background due to overloading of HRP-luminol causing increased enzyme activity. It is recommended that when you use high-sensitivity ECL, the primary and secondary antibody concentrations should be reduced. It is advisable to follow the manufacturer recommended antibody concentrations, if provided.

If you incorporate these measures in your protocol, you may be able to improve the sensitivity of Western Blot for your proteins having low signal levels.

Best.

Thank you, Sam Siljee , Vivek Kumar , and Malcolm Nobre , for your insightful contributions.

Sam Siljee Regarding the detection of Zo-1, I've observed a stark difference between brain cells and colon cell lines. While Zo-1 is readily detected in brain cells, its detection in colon cell lines has proven to be challenging.

Vivek Kumar I've employed Actin and GAPDH as reference genes. During Ponceau red staining after overnight transfer at 45V for 13 hours, I noticed intense red bands below 100 kDa, whereas faint bands were observed after 200 kDa. I will take your suggestion of dot blot into consideration.

Malcolm Nobre I've been using Ripa buffer containing 4% protease inhibitors and 1% phosphatase inhibitors for lysis. Interestingly, the low signal proteins I'm detecting include components of tight junctions like Zo-1, along with plasma membrane transporters/exchangers. Despite using a PVDF membrane (0.4 μm) and conducting overnight wet transfers at 45V, I'm encountering difficulties. I've adjusted antibody dilution based on initial recommendations and detection outcomes. Additionally, I've employed highly sensitive substrates such as ECL Amersham and West Femto, yet none have yielded positive results.

At this juncture, it seems further troubleshooting at the technical level may be necessary, or perhaps western blotting isn't the optimal method for detecting these proteins.

I would appreciate any suggestions offered.

I have seen people equilibrate the gel in transfer buffer for 30 min before transferring. If you do that as well then you might be loosing some large wt. proteins by diffusion.

Have you noticed insufficient transfer for larger proteins by staining the gel after the transfer with comassie blue?

More so often antibodies are the culprit, make sure to try first with the antibodies mentioned in the previously published papers, then refine.

Caco-2 cells can get decent banding for ZO-1 see figure (https://www.thermofisher.com/antibody/product/ZO-1-Antibody-clone-ZO1-1A12-Monoclonal/33-9100?gclid=CjwKCAiAuNGuBhAkEiwAGId4avF3g6-n9XEFuLaXX5Y1CF6xe1iEuTcWg7EpDkm01wN4jH_eIE4wLxoCsKcQAvD_BwE&ef_id=CjwKCAiAuNGuBhAkEiwAGId4avF3g6-n9XEFuLaXX5Y1CF6xe1iEuTcWg7EpDkm01wN4jH_eIE4wLxoCsKcQAvD_BwE:G:s&s_kwcid=AL!3652!3!459737518508!!!g!!!10950825775!106531320406&cid=bid_pca_aup_r01_co_cp1359_pjt0000_bid00000_0se_gaw_dy_pur_con&gad_source=1). Western blotting is a nuanced method, I feel your frustration but my guess is that with more optimization you probably can get it to work like the antibody above.

I appreciate your thoughtful response. I'll dedicate effort to optimizing the situation and remain optimistic about the potential results.