Dear all,
I have planned to study differential expression of proteins through 2-D electrophoresis (not any target protein) in 7 days old cotton seedlings under different abiotic stress (NaCl, PEG, Heat and Cold) conditions. I have planned to give stress treatment to isolated seedlings in double distilled water containing the stress inducing chemicals for 1hour and 12 hour duration. Is this the correct way to give stress treatment and is this exposure time enough to get protein expression or i should give more time? (i have only two more months to finish my phd work). Experts please help me to design this work correctly.
You can check Vyroubalová et al (2009) Characterization of New Maize Genes Putatively Involved in Cytokinin Metabolism and Their Expression during Osmotic Stress in Relation to Cytokinin Levels
http://www.plantphysiol.org/content/151/1/433.long
(I can send you the pdf, if you couldn't access it)
There we have stressed maize plants for 0.5 hour and 3 days.
Dear Sir,
Thank you very much. Already I have number of papers like this with me. All of them are related to gene expression quantification through qPCR. But my aim is to study the protein expression through 2D electrophoresis. My question is stress treatment for 1h and 12 h enough for getting expression of stress related proteins or i should give stress in days?
I see, sorry about that.
In that case 1 and 3 hours should follow the early response. E.g. here http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4393234/ they do histochemical analysis during first 12 hours after treatment.
dear Prem,
Wish to share a few thoughts regarding your protein expression query:
1. The stress induction in seedlings occurs in short period, its means we can study the protein profile.
2. For above supporting data hope you’re done a few ROS related assay (in time kinetics) in your previous studies, which is a clue for present work plan regarding the time: 1hr and 12hr.
3. But the expression of proteins to abiotic stress depends on factors :
A. Concentration & dosage of inducer
B. Incubation condition: temperature, humidity, etc.,
C. Time interval
D. Type of seedlings/ seed variety
4. Since you are planned for 2month it’s seems appropriate to opt 4r single concentration study with different stress inducer and one set of control (simple with effective data)
5. If protein extraction protocol is already standardized for this particular work then it’s easier or else takes up your time.
6. Most important is to concentrate on preparation of the samples and other required chemical/solutions (a single mistake it takes up lot of time)
7. If all works out well it requires max 3months with analysis & interpretation of data.
All the best
Hi Bhavya,
Thanks for your positive suggestion. Already i standardized protein extraction protocol for the plant. The one and only doubt i had was duration of stress treatment. Now im clear. Thank you once again.
The duration of stress treatment depends on whether you want to see the first response (1 or 3 hours) or the acclimation (24 hours and more).
Dear Sir,
i want to look for difference in protein profile in response to different stress at two time frames (1h and 12 h).
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