help for the method of meat sample preperation for SDS PAGE 1D - (Mar/27/2012 )

Hello everybody,
I have meat protein samples prepared according to Laemmli as following;

2,5 g of meat sample + 25 ml of Tampon I (tris,sucrose,EDTA)
....centrifugation....(1000g 10 min)
...trowing of supernatant...
precipitated material + 25 ml of Tampon II (Tris, EDTA)
....centrifugation....(1000g 10 min)
...trowing of supernatant...
precipitated material + 25 ml of KCl (0,15 M)
....centrifugation....(1000g 10 min)
...trowing of supernatant...
precipitated material + 25 ml of KCl (0,15 M)
....centrifugation....(1000g 10 min)
...trowing of supernatant...
lyofilization of precipitated material.

Here i want to ask you is that,
May i use the same protocol to prepare the samples ready to load into the gel which is used for 2D SDS-PAGE and as described below?
and after the last step what will i do to make the samples ready to load on sds-page?
..weight 0,03 g of lyofilized sample in a 2 ml eppis
500 microliter lysisbuffer
32,5 microliter DDT
20 microliter phosphatase inhibitor
...wait 30 min in ice
...ultrasonic bath for 1,5 min.
...wait 10 min in the ice
...centrifuge at 20.000 g-4C-30 min
...collect supernatant in a 1,5 ml eppis
...use bradford to determine protein concentration
....??????

or do i have to use the protocol which we use water bath at 50 C for 1 h and wait for overnight than filter the solution than directly load on the 7 microliters of bromofenol blue in the wells????

My major concern with running a 2DE following your sample prep and lysis steps would be the ability for the isoelectric focuser to effectively focus your proteins of interest (since you have added EDTA, sucrose, KCl and whatever else might be in your lysis buffer). Depending on the amount of sample you end up with, you may want to look into doing some sort of a cleanup precipitation (e.g. acetonitrile or acetone), ultrafiltration or dialysis.

proteaMatt on Tue Mar 27 14:16:17 2012 said:

cilem is using the pellet, not the supernate, from each step. there may be some residual kcl in the pellet but that should be diluted by the lysis buffer. and he is not running ief, just sds-page.

what is the lysis buffer when preparing the sample for the first dimension?

the alternative protocol (50C, etc) should be okay if you are incubating in laemmli sample solution. keep in mind that residual kcl may cause precipitation of sds (actually, kds), so you may want to perform one or two washes with water before you lyophilize.

I guess I misunderstood the OP, somewhere I got it in my mind he was trying to prep for 2DE.

proteaMatt on Wed Mar 28 15:10:48 2012 said:

i thought the same, at first. i reread the question and realized that the op was just using the preparation technique for 2d but was only going on to sds-page.

@cilem: if you prepare the sample as you would for 2d then, after the bradford, you would just add laemmli sample buffer to the sample, boil and load the gel (assuming that the lysis buffer is compatible, otherwise you may have to precipitate the protein away from the buffer before adding sample buffer).