I performed 2D- GE of bacterial culture Cellulosimicrobium cellulans under control and treated conditions. I have loaded 1.2 mg sample on pH strips 4-7 (13 cm). Everytime I get this streaking as well as clustered protein spots at the acidic end. I have solubilized protein in rehydration buffer with urea and thiourea. Please suggest necessary steps to avoid streaking and better focusing of the protein.
Hello,
To have good resolution and reproducibility on 2D gels is always tricky. I don't have all the details of your protocol but as you have both horizontal and vertical streaking you could start with some basic and simple steps on both dimensions and see if this improves (see the points below for some ideas). You might also consider loading less amount of sample per gel (eg. 100 ug).
- you may change the pH range of the strips aiming at better resolved the majority of acidic proteins in your samples (I believe there are some 3-6 pH strips)
- you may use ampholyte (an adequate amount and type)
- you may incorporate a 5h passive rehydration step (at 20°C) before launching the active rehydration step
- you should optimize the isoelectric focusing avoiding a fast increase of the voltage ramping and considering that IEF higher than 10,000 V.h could lead to horizontal streaking but also too low Vh.
- you may leave the gels polymerizing for at least 8h (to avoid dessecation, when the linear phase has been formed with butanol, discard it, wash extensively and replace by water)
- For protein migration in the gels you can also launch a first program of 25 mA/gel for 30 min to allow the samples to slowly enter into the gel and then increase the voltage for protein separation (e.g 75 mA/gel).
Hope this help!
Annia
Thankyou Annia Alba ..for your suggestions..I'll like to defend myself on the points you mentioned;
- I have used ampholyte (IPG Buffer) 0.2% v/v in the rehydration buffer.
- I provide passive rehydration for 15 hr at room temp and then proceed with IEF.
- I am attaching a file for IEF procedure.
-For protein separation, I initially go with 50 V/gel for 1hr and then 100V/gel for 3-4 hr.
Rest of the suggestions I'll take into account.
Feel free to rectify me if I'm proceeding with any mistakes.
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