There exists ImmunoPCR and its different versions (such as TaqMan protein assays) but it still requires antibodies and you will have tons of trouble with it, much, much more than if you just did Western. And it will be much more expensive. Why don't you use blotting for quantification? It is a standard, established technique, you should have no trouble publishing results.

Western blot can give you information on relative protein amount but is not traditionally considered a 'quantitative' technique. If you want data that's a bit more quantitative, you can use qRT-PCR. Remember though, qRT-PCR is quantifying the amount of specific mRNA species in your sample. So, you can infer protein amount by using this method, but remember.. just because the mRNA is present at a certain level does not guarantee an equally proportionate amount of the corresponding protein is also present.

For qRT-PCR you will need some materials that are unique to this method. The thermal-cycler that is used is a bit different from the traditional machines used in most PCR applications. Check if your department has this equipment. Then you will need to find the appropriate reagents to prepare your reactions. I strongly advise you run each reaction in triplicate (duplicate, at a minimum). I have used the BioRad reagents in the past and it worked well. Thermo also has many products that will work.

Finally, you need to design primers that are complimentary to the mRNA transcript you are probing for. There are online guides available to help with this (e.g. https://bitesizebio.com/10041/designing-qpcr-primers/ )

In case you're not clear about how this process works - qRT-PCR is a method that uses PCR amplification to check for the amount of specific mRNA transcripts in your sample. To do this, you first need to isolate the RNA in your sample. Then the mRNA in your sample must be converted to cDNA (complimentary DNA). Then you can probe the cDNA using primers you have designed. In simple terms, this method will count the number of amplification cycles for each probe. By comparing the number of cycles it takes to amplify different targets (and comparing to a "housekeeping"/reference gene, e.g. GAPDH, actin, 18S, etc), you can quantify the expression level of your target genes.

Workflow summary:

Sample > isolate RNA > cDNA > qRT-PCR

Hi there,

qRT-PCR is only measuring mRNA amount. Which is not indicative of the actual level of the corresponding protein.

Hi, Nidhi Rajput

I suppose you're interested in qPCR for protein detection. Am I right? If so, you can take a look at some companies websites like (I am not recommending any company products with my comment. Ok? Only data for your own evaluation):

https://www.thermofisher.com/br/en/home/life-science/pcr/real-time-pcr/real-time-pcr-applications/real-time-pcr-protein-analysis.html

https://www.thermofisher.com/br/en/home/life-science/pcr/real-time-pcr/real-time-pcr-applications/real-time-pcr-protein-analysis/protein-thermal-shift.html

https://lifescience.roche.com/en_br/blog/lab-life/high-throughput/important-factors-to-consider-for-thermal-shift-assays.html

https://biotium.com/product/glomelt-thermal-shift-protein-stability-kit/

https://cmi.hms.harvard.edu/differential-scanning-fluorimetry

Even a protocol can be found here from a Harvard team using SYPRO Orange:

http://www.its.caltech.edu/~bjorker/thermofluor_assay.pdf

https://www.transcriptic.com/technical-notes/thermal-shift/

If I am correct, these will help you to think about it.

Hope it helps.

Hi Nidhi,

To begin,

you must treat the cells as per your conditions and then collect the cells at the expected time point.

Extract the RNA from the cells.

Prepare cDNA from the kit that is available with you. (you may use 2000ng of RNA to ma cDNA)

I generally dilute the cDNA to 1:1 ratio with water. I use 2 ul for my reaction.

Next, I use Sybr Green kit (Invitrogen) to make qPCR reactions, load the plate and run the program. In 2 hours, I get the result. Now, here you need to run a control as well along with standards. I use B-Actin, GAPDH in general experiments. Check the literature for your specific protein.

Then, analyse your results. For further queries, write back. Good luck.

Pratibha

I think qPCR can only tell you about the mRNA level at particular point but not the protein.

Hi to you all.

Yes, it is possible to perform protein quantitation by qPCR. But I am not sure If it is what Nidhi Rajput really wants. I am waiting for her answer in order to know it.

Links in my last response show it.

All the best.

Adriano Costa de Alcantara Thank you sir, this is was i am actually looking for to quantifu proteins using qPCR. The links you have shared are helpful, thanks for sharing.

Michael Connelly, thank you for the information.