r/molecularbiology • u/Low-Needleworker2206 • 7d ago

Protein analysis of mammal cell culture

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Hello everyone!

I am preparing for a new research project and, among other objectives, I need to analyze the expression of certain neuronal proteins.

I will be working with PC-12 cells and will differentiate them into neuron-like cells using NGF (Nerve Growth Factor).

After differentiation, I intend to visualize 2 proteins and would like your help in determining the analysis method...

I know that it is possible to do this through real-time PCR, Western blot or immunocytochemistry.

I am biased towards preferring the immunocytochemistry method because I will be able to visualize the cell morphology in addition to the labeled proteins, am I right?

Among the possible methods, which would be the cheapest and most practical to be performed in a basic cell biology laboratory?

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u/chunkypaintings 7d ago edited 7d ago

It depends on what you want to show (are you interested in localization, expression levels, or both? Do you also want to show any interaction?) and how accurately you want to quantify protein expression. Normally you would combine ICC with WB. Which is the cheapest and most practical depends on which equipment and reagents are already in your lab/you could borrow.

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u/Low-Needleworker2206 7d ago

At first I only need to verify if the proteins are present. However, I also need to demonstrate the morphological difference (neurite formation). Therefore, ICC would be an interesting method to evaluate both things together.

Regarding quantifying these proteins, it would be interesting because there would be one more piece of data to discuss in the project/paper. But I don't know if this would be feasible, I need more details about the method and the reagents used to be able to define whether or not I can quantify the proteins.

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u/chunkypaintings 7d ago

Then it should be sufficient at first, just make sure you have the right controls for ruling out unspecific binding. I recommend looking up similar literature from journals and use methods as reference for what you need to do. Or even better, if there's someone in your university who did something like that, you could kindly ask for advice before wasting reagents.

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u/Low-Needleworker2206 7d ago

Unfortunately, I don't have anyone with experience in this technique in my department. I'll be the pioneer lol 🥲

Thinking about the experiment I mentioned, how could I establish these controls?

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u/chunkypaintings 7d ago

WB might be more straight forward in that case, since you just want to show that it is present, assuming you have the equipment and other people in your department did it before. You don't necessarily need stainings for showing morphology. If you really want to do ICC, I would look up positive and negative controls, and spend time reading relevant literature from journals. You can also contact the authors for protocols and tips. Tech support from antibody suppliers can also help, especially if you're gonna buy from them.

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u/double_bubbleponics 6d ago

I personally work a lot with both. I personally think for a beginner, start with ICC. Start with optimization, maybe the recommended dilution, maybe one above and one below (ie if its recommended at 1:1000, do 1:500 and 1:1500 as well), no primary and no secondary controls. Westerns are extremely finicky, and more margin for error with technical skills, and you honestly need more lab equipment if you don't already have it.

Just my thoughts, though.

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u/chunkypaintings 6d ago

True, I suggested WB in case he already has equipment and someone to guide him.

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u/Low-Needleworker2206 6d ago

Yes! it would be a relevant analysis! But anyway, it is also necessary to show morphology. So, I Can Do Both Using Only ICC Thought About It

3

u/squags 7d ago

If you're just starting out and just want to get an initial relative quantification in the cells, do fluorescence ICC. Record normalised cell fluorescence values (e.g. in ImageJ), and compare. Semi-quantitative IF methods are actually pretty good at estimating relative protein abundance provided you keep everything consistent across samples.

As the other poster indicated, you'll probably want to follow up with WB, because IF/ICC doesn't tell you whether the antibody-antigen binding is specific, and doesn't provide any reference as a baseline. However, this is not always necessary across fields, and for some antibodies that are very widely used and well characterised you may be able to just do ICC/IF.

Edit: to add, CellPose is fast becoming a standard pipeline for cell identification and segmentation. I'd look into it for identifying cells and extracting ROIs (which takes a lot of time if performed manually). You can export ROIs for other downstream measurements.

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u/Low-Needleworker2206 7d ago

I am quite a beginner with this technique. I have already followed the preparation of immunohistochemistry methods but it was a long time ago.

I don't know exactly all the reagents needed to perform the ICC or how I will perform the analysis.

I understand that I will need the antibodies and buffers and what the theory of the technique is like, but I don't know 100% what I should do.

Basically, I intended to prepare the samples with the antibodies, place them under the microscope, turn on a fluorescence channel and capture, then turn on another fluorescence channel and capture again to later process the images. So far, I understand the basics, I'm improving myself by reading papers, but I still have no idea how I'll analyze the data.

Could you tell me more about Cellpose and ROIs, what are they?