I treat cells then compare protein expression compared to untreated. If I test five protein, do I need to seed in five well of six well plate or just one.
As a general answer, you should have one well for untreated cells and one for treated. Then make a cell lysate from each, run both samples on the same gel and blot for each protein. You should also blot for at least one loading control such as actin (and make sure your are loading the same quantity of protein and/or cell equivalents per lane). However, a reason to use more wells would be to do replicates. If possible, I would do the entire experiment over at least three times. If not, then you should consider triplicate wells of both treated and untreated on the same occasion. Another possible reason is to get more lysate if you might run out before you run all your blots (and it might be easier to use two 10 cm dishes instead in this case). You will learn how many cells you really need for the samples based on experience with your particular assays.
Hi there,
Well it depends on the detection : do you use the same antibody for the whole set of proteins (which means they are all tagged) or do you use different antibodies? If the former then proteins have to exhibit different mobilities on gel in order to identify them individually. If the latter then each protein needs a specific probing so you need one lane per sample and per probing.
Dear Heba,
You can test all proteins in one well. But they should not be close in size. I think you should use high-quality monoclonal antibody. So you can prevent non-specific antibody binding.
Hello,
I do triplicate of each condition + house keeping control+ positive. Then I used to run all the available samples and repeat that three times for each preotein.
Dear Heba, I agree with the Ozen answers; five proteins are a lot, but with monoclonal antibodies you can choose the protein not close in size and perform the incubation together. You can try, for example, three antibodies and see how clean are the results. You have to pay attention to the different expression, some protein could be more expressed than other (pay attention to secondary dilution) and you can decide to cut the nitrocellulose (or PVDF) membranes to improve the results (in particular for the loading control).
For the replicates I think that it would be better to perform three technical replicates (treated and untreated cells) and two/ three biological replicates for your experiment
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