Hi, you are confused because you studied hard, you used your co-worker protocols or made researches on the web and then you did not know what to do. I think that the problems is that you do not ask the correct question. What do you want to analyse? You said, “I am presently planning to do western blot”, ok of what? What proteins do you want to study in your cells? Briefly what is your project goal?

Do you have to analysed cytoplasmatic or membrane cells (plasmatic, nuclear)? Do you have to perform immunoprecipitation? Do you have to study protein interactions?

I ask you this because there are many different lysis buffers for this reason. Your co-workers used samples different from yours, maybe they study proteins that were difficult to solubilized, easily degraded and have different recipes. When I have doubts about the buffers I used pub med to find works that analysed my protein of interest, preferable in same samples. It is normal to find recipes with some difference (sometimes same reagent but different concentration), because it is important optimized the buffers following your experimental condition.

There are many sites where you can find a good general description about lysis buffers component (as a lot of researchgate discussion about the topic)

http://www.abcam.com/protocols/sample-preparation-for-western-blot

https://advansta.com/detergent-lysis-buffer/

After that you have to find some information about the proteins you want to analyse. If for example you want to study a cytoplasmatic protein you can begin whit NP-40 buffer or RIPA buffer, you can choose also two types of buffers (with protease inhibitor), extract two H9C2 pellets (I used RIPA with this cells for membrane proteins) each with a different buffer and perform a Bradford assay.

Another important thing is what is the methods used in you lab to extract proteins? In general, with cells pellet you can sonicate the pellet or do you have a mini potter?

I hope I do not confused you even futher :)

Dear respective Professor, thank you so much for addition of your valuable answer. And, underneath, I briefly describe my project goal.

Project goal: The main focus of this project is to evaluate the cardioprotective effect of irisin against cardiac I/R injury. Briefly, mitochondrial mPTP (membrane permeability transition pore) opening, mitochondrial swelling are leading to cellular necroptosis. So, firstly I would like to estimate the RIPK1/3 level. After That, as we know from previous study, disruption of mitochondrial membrane is the major source of ROS generation which lead to cellular apoptosis via elevated caspase levels. So, then I would evaluate the pro-apoptotic protein level such as caspase-3. Finally, many studies already uncovered that autophagy is an intracellular process responsible for damaged or unnecessary protein and organelle degradation. In the heart, autophagy occurs at basal level and dysregulated autophagy is associated with a variety of cardiovascular diseases. Later on, I would like also calculate the level of Beclin-1, LC3, ATG-5, ATG-7, ATG-12 respectively.

Hi, first of all call me Valeria, I am not a Professor : )

I have not experience in autophagy or mitochondria biology but if I think that you would perform a treatment on your H9C2 so, at the end you will analyse treated (different concentrations?) and not treated samples. Is it correct?

Before preparing difficult experiments with many sample, you have to be sure to perform a good western with good signals for all of the proteins listed above. You have many different proteins to analysis, most of them are cytoplasmatic but other interact or bind to organelle membranes. In general you would like if it is possible, to analyses these proteins, or the ones that belong to a specific pathway, all together on a western, so you can analyses in a single experiment (that you will repeat) the different expression of your proteins of interest. I think that you have to:

· Reading a lot of literature about western on these proteins, preferably on your cell lines, to choose the best buffer, antibodies, to have advice on treatments, etc

· Choosing one or two lysis buffers that can help you to achieve this purpose and then optimized the conditions

· Paying attention, as I said before, if some of this proteins are temperature sensible and are easily degraded

· Testing first western blot with your pellet H9C2 (if the proteins are expressed at basal levels), or positive controls, to see if your antibodies are good. I write positive control because if you cannot detect a proteins in untread cells (basal levels) maybe the signal is too low and you have to use another cell lines as positive control.

These are only some examples or advices to begin with.

If you have antibodies for some of your proteins of interest in your lab, and you know that you can detect a signal in untreated H9C2, you can begin to extract proteins from your pellet. Ask advice to your co-worker for extraction method (proteins extraction from cell line is, in general, more easy that from tissue), and for measure of sample protein concentration. As I said before use different buffers, prepare two, four samples and begin to “adventure in the land of western blot”.

It is not easy and you will face probably some challenges, do not be discouraged, we all went through it!

Thanks Valeria, and you prediction is absolutely correct, that at the end I will analyse treated (different concentrations of irisin) and not treated samples. Thereafter, I would like to inform you that, I also have a plan to explore specific signaling pathway along with particular proteins.

Dear Valeria, I already guessed, you are not only a astounding researcher but also you have a wonderful skill as well as mentality for knowledge sharing. And, I am really blessed for your most valuable advice. I strongly believe your recommendation will significantly assist my upcoming experiments. Finally, I hope I will well address your advice to my experiments.

Dear Valeria, if you have no problem, could you please provide me your western blot protocol along with troubleshooting guide?

Thanks you are very kind, I am not absolutely astounding researcher, but I like to share and help if I can because I know the meaning to work wihout advices and the meaning to be help by very astounding researchers.

I add some files, at the beginning you will be overwhelmed by all the information, take your time, one step at a time. Begin with extraction and measure of protein concentration, read only the files or protocols o datasheets that you need, be sure to prepare good samples, then move to SDS-page and western blot.

Study the principles why you do this or that, be aware, be informed, it is true for all the things that you would do in the future.

I suggest you to refer to this community (or other) for advices, I always find help here and very kind people. If you want ask me something, send me private messages, I would be happy to help you.