Dear coleagues,
I need to analyse total protein content in Arabidopsis leaves and I have very less leaf material.
I haven`t find so many works where, specifically, protein content was analysed usually they were doing imunoblot analysis or gel electrophoresis and I am not interested in this data. I do not know how much of total extracted protein concentration I can expect from ca. 50 mg of leaf material.
I found inforamation that total protein conten from the plant at the 4 day stage was in a range between 0.5- 10 micrograms/ microlitar or 5 - 10 micrograms / microlitar for 3 weeks adult plants.
In my case I am using leaves of 4 weeks old Arabidopsisi plant and the weight of mz samples are ca. 100 mg.
Thank you very much in advance!
S.
you could use the method of Lowery for the determination of total soluble proteins.
Here is a good general overview of the methods you could use (http://people.umass.edu/~mcclemen/581Proteins.html). You should consider what you want to do with the protein as well, if you want to extract the proteins for enzyme analysis, for immunoblot analysis, or are just interested in total (soluble?) protein content. I imagine the variances in protein content you cite could be due to genotype, growth conditions (soil vs agar, fertilization, light regime, temperature, watering schedule which would change fresh weight), *not to mention extraction efficiency* (how good a job you do at extracting the proteins out of the cellular matrix), and inclusion of protein solubilizing reagents (Triton X100, Nonidet P40, thiourea etc).
The strategy I would recommend depends on what your end goal is. In other words, what are you doing with the protein after you've extracted it? (Or are you just looking at at gross protein content?) What information do you ultimately want?
Cold Spring Harbor has a protocol (written by Rob Larkin in 2007; see link below) which is a good starting point that uses relatively simple methods (Bradford) and inexpensive reagents. I've also used a kit from Sigma-Aldrich with good results, but it's much more costly.
As for total protein content, your estimate of 5-10ug/uL is reasonable, but it varies considerably depending on accession and growth conditions. Speaking from personal experience, I have gotten more out of 4-week-old Col-0 whole plant samples grown under benign, long day, growth room conditions, but that may not apply to your experiment.
Puno sreće!
http://cshprotocols.cshlp.org/content/2007/1/pdb.prot4680.full
Anyway in 50-100 mg tissue you should have plenty enough material for any application i suppose
Depends on what you what to do with the total proteins extracted. From 50-100 mg leaf tissue, you will get more than enough proteins for a LC-MS analysis. I have used the Sigma's Total plant protein kit which is a bit expensive, but with very good protein quality for LC-MS. CSHL protocols are also very good for homemade extraction protocols.
Bradford is also my first thought, but include as many technical replicates as possible
Hi Sladjana,
Usually I had got 10 ug per 6 weeks-old Arabidopsis leaf (20mg) extracting total protein with SDS- extraction buffer to western blot, but I'm not sure if you can use this kind of buffer. If this is your case, there are other protocols where you can extract soluble and membrane/wall proteins that I attached. You can determined protein content by bradford or Lowry but should be in a similar magnitud order than others (may be little bit less, since there are not SDS or other detergent)
Please find attached two different methods to extract proteins. The last one is the traditional with SDS.
All the best!
http://www.scigine.com/method.php?ID=147001
http://www.scigine.com/method.php?ID=190596
http://www.scigine.com/method.php?ID=218829
Hi I want to use the Lowry method for protein quantification in soybean leaf samples. Can anybody please guide me from the beginning like grinding of leaf samples in buffer. I read many papers. Authors use different centrifuge. I want to use maximum of 14000 rpm benchtop. Also not temperature controlled. I even don't want the fancy MS. I just want to measure through spectrophotometer the TSP total soluble proteins. Please guide me
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