Hi, I´m a new researcher and I am trying to verify a particular cell line before I start experiments. I am looking at expression of certain key proteins specific to this cell line.
I have been having trouble detecting all these proteins by western blot- getting either faint bands or no bands at all- partly because of my proficiency.
I am quite keen to know if immunofluorescence staining of these 3 key proteins would be sufficient to achieve this similar goal? that is, if there is immunostaining seen, will that be enough?
Very few proteins are unique to a specific cell lines. Also, very few antibodies are absolutely specific, either in IF or Western blotting. The fact that you are not getting the appropriate signals may be due to your proficiency, as you say, or it may be that the proteins are just not there. If you are questioning your proficiency, then even a positive result would be questionable, wouldn't it? False positives in IF and misidentified bands in Western blotting are very common. Therefore, clearly this is not an acceptable method of cell line authentication. The most accepted method of authenticating cell lines (and authentication should always be done, especially if the cells were obtained from another lab, rather than from e.g. ATCC) is STR-based DNA fingerprinting. You can do this yourself, or have it done as a service if you are not familiar with the techniques. See for example,
https://www.promega.ca/resources/pubhub/cell-line-authentication-with-strs-2012-update/
and also look at https://www.atcc.org/~/media/PDFs/Technical%20Bulletins/tb08.ashx
If you are planning to spend a significant part of your career working with a particular cell line, it is well worth the effort to properly authenticate them. It could save you a lot of time, money and embarrassment.
Hello Iwan,
IF is a standard criterion to characterize cell lines and then can be opted well in this case. The reason for faint bands on WB may be due to handling errors as you have said and sometime in ab also..like same ab which is suitable for IF may not work fr WB. But with IF I will suggest you to run a negative control always (to avoid the possiblity of non specific staining/autofluorescence) and you have to take care that proteins you are looking for, are exclusively expressed by those cells.
Hope it helps !
Very few proteins are unique to a specific cell lines. Also, very few antibodies are absolutely specific, either in IF or Western blotting. The fact that you are not getting the appropriate signals may be due to your proficiency, as you say, or it may be that the proteins are just not there. If you are questioning your proficiency, then even a positive result would be questionable, wouldn't it? False positives in IF and misidentified bands in Western blotting are very common. Therefore, clearly this is not an acceptable method of cell line authentication. The most accepted method of authenticating cell lines (and authentication should always be done, especially if the cells were obtained from another lab, rather than from e.g. ATCC) is STR-based DNA fingerprinting. You can do this yourself, or have it done as a service if you are not familiar with the techniques. See for example,
https://www.promega.ca/resources/pubhub/cell-line-authentication-with-strs-2012-update/
and also look at https://www.atcc.org/~/media/PDFs/Technical%20Bulletins/tb08.ashx
If you are planning to spend a significant part of your career working with a particular cell line, it is well worth the effort to properly authenticate them. It could save you a lot of time, money and embarrassment.
Hi Iwan. I wanted to add to the conversation by saying that if you are using a non-human cell line (e.g. mouse cell line like RAW 264.7), our lab usually sends out samples to IDEXX. They are a company that specializes in non-human cell line authentication. Here's a link: www.idexxbioresearch.com
Also, I'm attaching a paper that helped me understand new cell line authentication standards. I hope this helps and good luck in your research!
-Bryan
www.idexxbioresearch.com
Hello Iwan, if I understand your questions correctly. You wanted to test several cell lines to check which cell lines have highly expressed of your target protein right? You've already known the cell lines you are using. You tested them by western blot and IF.
If you have trouble with Westernblot, you can compare the expression level of that protein by IF. However, WB will give you a better answer than IF because it has a control so you can compare the expression level of target protein in different cell lines.
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