I am trying to detect a secreted protein by Western blot in plan tissue. To this aim, we are using a simple protein extraction protocol based on TX-100 and DTT. Then, I am loading the samples in a SDS-PAGE and doing western blotting. However, I don´t detect my protein eventhough it should be overexpressed… We have read that this protein is secreted. Should I perform another protocol to identify it? Can this protocol somehow promote degradation of my protein ?
Secreted proteins should be detectable by Western blot. Depending on secretion efficiency, you might get a low amount in the media because the majority of proteins could be stuck to the cell wall if they are non-native.
To increase sensitivity, you can consider ELISA, increase your protein concentration before WB by concentrating with a spin column or precipitating the protein, or use a different brand of proteinase inhibitor cocktails to reduce degradation.
If you have the protein or DNA sequence, maybe also check for the secretory sequence with an online server (google 'signal peptide prediction' and choose a suitable host).
I am not sure if protein components are involved in plant culture media, but for mammalian cells, the presence of FBS (part of the essential media) might interfere with detection by overloading your SDS gels.
Good luck!
Plant tissues often contain polyphenol oxidase enzymes that can damage proteins by chemical modification. To avoid this, include polyvinylpyrrolidone in the extraction buffer. Also include protease inhibitor cocktail.
Leran Mao Thanks for your kind answer! we will try to increase protein concentration. By any change do you know any way to extract only the secreted proteins to enrich our protein of interest? We are not working in vitro but in vivo. Thanks again!
Álvaro Sánchez
Glad to help!
I'm not very sure what you mean by 'working in vivo'. Considering your proteins are secreted? Typically we just use a centrifuge to spin down cells and debris to collect the media and secreted proteins. Then clarify it with a 0.2 uM filter before purification.
Unfortunately I don't think there is a good way to do that and you usually would end up with some host cell proteins. We sometimes could purify our protein of interest (usually if it's expressed at large amounts compared to host proteins) via affinity /size or ionic exchange chromatography (or a combination thereof) - but only if you have a tag to the protein of interest or if it differs in physical characterisics pretty significantly from your host cell proteins (you can look up the pI of the protein).
Possibly you can choose a MWCO filter to filter out smaller proteins and enrich your protein of interest. If you decide there is not a large amount of host cell proteins (via Coomassie staining or likewise), you can consider purifying your protein via chromatography - note that you can only do this if you have a large batch to begin with (>100 mL media).
Another way is to do an immunoprecipitation and purify your protein with a specific antibody - but you might get IgG contamination in your solution too.
If you just want to confirm and quantify your protein is secreted, an ELISA usually works pretty well.
Good luck!
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