I am looking for the protein expression of Methanosarcina mazeii that can get expressed in the presence of acetate. I am not much aware of steps. I was planning to do cell-lysis and then SDS-PAGE to check the protein expression. What other steps I should take to do this? And can we link the protein expression with ant metabolic pathway? Any suggestion would be really helpful.
Terms mentioned by you are wrong.
When gene expression comes, researchers use RNA, real time PCR and etc.
When proteins expression comes, researchers use sds page methods.
Now what do you do exactly?
Regards
If you want to analyze changes in the general pattern of proteins, you should go for 2-D gel analysis (isoelectric focusing +SDS-PAGE).You will no doubt find plenty of protocols on the Internet. SDS-PAGE by itself is not sufficient to resolve all protein components present in a cell.
If you are interested in RNA expression, it can be performed by
1. Real time PCR: the level of mRNA are quantified by reverse transcription of RNA to cDNA followed by quantitative PCR of cDNA
2. Northern blotting: the levels of mRNA are directly quantitated by electrophoresis and transfer to membrane followed by incubation with specific probes.
If you are interested in protein expression, it can be performed by:
1. SDS-PAGE: Electrophoretically separation of proteins by size/mass, followed by protein staining with dyes such as Coomassie blue/silver staining or other dyes. Molecular weight standards should be run in parallel to know the molecular weight of each protein band visible in SDS-PAGE gel.
2. 2-D gel electrophoresis: It combines isoelectric focusing and SDS PAGE. Herein the protein expression is achieved by separating the extracted protein in two dimensions (first dimension is IEF (Separation based on pH) and 2nd dimension is SDS PAGE (based on mass). This is followed by staining to detect differences at the whole proteome level.
3. Mass spectrometry: For further identification of differentially expressed proteins (obtained in 2-D PAGE) and to know the structural details, LC-MS/MS is performed wherein the proteins are identified based on their mass.
4. Western blotting: quantification of relative expression of specific proteins is accomplished by electrophoretically separating extracted proteins, transferring them onto a membrane and then incubating with specific antibodies (targeted to specific protein of interest) that is subsequently detected using various detection methods.
5. Immunoassays: Protein are quantitated using antibodies and is detected by chromogenic or fluorogenic substrates.
For general expression pattern of all the proteins, SDS-PAGE or 2DE can be used depending on the level of separation of proteins you need.
Hello Aliraza,
I am sorry about the confusion. I am looking forward for the protein expression and their identification.
Thankyou everyone for the suggestions. Thanks Pierre. Thankyou Bhairavi for the detailed answer, I will follow the steps you mentioned to identify and characterize the protein.
Now if you're doing a forward approach Pierre's answer is perfect; run a 2-D gel, do proper controls, probably run a 2D DIGE eventually. If your lab is properly funded, then you could do a whole-proteomic approach; simply isolate all the proteins and MS-spec the crap out of everything. If you already have a target pathway in mind, then you could do a reverse approach; buy antibodies against all (or key) proteins in that pathway, and just run western blots for the rest of your study.
Hi, Priyanka Srivastava.
Just to give you some free/open manual that may give you some good background in the subject you may look at some known companies and their manuals, such as qiagen, GE, Thermo ...
1 - https://www.qiagen.com/mx/resources/resourcedetail?id=79ca2f7d-42fe-4d62-8676-4cfa948c9435&lang=en
2 - https://cdn.gelifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=13891
3 - https://www.thermofisher.com/content/dam/LifeTech/global/Forms/PDF/protein-expression-handbook.pdf
I hope this nice basic manuals may help you.
All the best.
P.S. May I ask you If you have any relative called Pramod K. Srivastava? If so, send him my regards (lol).
In any case, you need to have both, cytosols from your experimental with acetate and cytosol prepared from controls grown in all respects identical, but without acetate.
If you are looking for changes in the expression of specific enzymes, then comparison of their activity (say, in nkat/million cells) is the most specific and easiest to perform method.
If, however, you look for general changes in protein expression patterns, differential 2D-electrophoresis (DIGE) is probably easiest. In DIGE, proteins are labelled with fluorescent dye before electrophoresis, with a dye that doesn't change the charge of the proteins. You use, say, a green dye for your controls and a red one for your experimental. Then a protein whose expression is unchanged will result in yellow spots (mix of red and green). If, however, expression of a protein is increased in the experimental, spots will appear red, if it is decreased, green. See for example doi:10.1021/bc00020a001, doi:10.1002/elps.1150181133, doi:10.1093/brain/awg208 and doi:10.1038/13690
could do, analysis of proteomics with mass spectrometry, using LFQ (label free quantification), using offline fractionation strategies.
1. As a first step, extract the proteins with a lysis buffer.
2. then clean up the samples with chloroform / methanol.
3. Resuspend with guanidinium 6M, reduce with DTT, alquilation with AMI, and digest with trypsin.
If you need help with the protocols, you can write to [email protected]
Thankyou Engelbert. Thankyou Mauricio. I think I will be going with LFQ.
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