9 replies to this topic

[RanjnaSCheema]

I am working on proteins of dog spermatozoa and trying to separate sperm membrane proteins with 2-D gel electrophoresis. I first tried 2-D of SDS-sperm membrane extracts, cleaned with 2-D clean up kit of Biorad, but did not get any spots. I also tried to extarct proteins directly in Multiple surfactant solution of Biorad i.e. 5M urea, 2M Thiourea, 2% CHAPS, 1% DTT,, 2mM TBP,2% SB-3-10, 0.2% carrier ampholytes, 40 mM Tris, but even then did not get any spots, but just get a line near the dye front. The composition of equilibration buffer  used is 6M urea, 2% DTT, 1% SDS, 2% CHAPS, 40 mM Tris, pH 8.8. I have been working on these protocols by doing one or the other modifications for the last six months, but did not get any satisfactory results. Kindly help me to sort out this problem, as I want proper separation in 2-D gels for my project work.

[mdfenko]

have you confirmed separation in the first dimension?

does your first dimension also contain urea, etc?

is your protein solubilized in the urea solutions? clarified of insoluble material?

[proteaMatt]

What focusing conditions are you operating under? When I focus serum samples or lysates I typically have to increase the amount of volt hours for focusing because all the salts will inhibit focusing. I assume you are using gel strips. Have you tried using linear vs. non-linear pH ranges or different pH ranges altogether?

[RanjnaSCheema]

mdfenko, on 27 February 2012 - 08:48 AM, said:

have you confirmed separation in the first dimension?

does your first dimension also contain urea, etc?

is your protein solubilized in the urea solutions? clarified of insoluble material?

Yes, I confirmed separation in the first dimension. I do SDS-PAGE of SDS-extracts. But for 2-D, I solubilize sperm proteins directly in rehydration buffer. Before, rehdrationof IPG strip, I do centrifuge my sample at 10,000g for 10 min. Sometimes, I do get very few spots and streks in the lower part of gel.

[RanjnaSCheema]

proteaMatt, on 28 February 2012 - 07:24 AM, said:

What focusing conditions are you operating under? When I focus serum samples or lysates I typically have to increase the amount of volt hours for focusing because all the salts will inhibit focusing. I assume you are using gel strips. Have you tried using linear vs. non-linear pH ranges or different pH ranges altogether?

Focussing conditions are as per the guidelines of CLEVERS equipment
Volts     Time
150   30 min
300   30 min
600   30 min
1500     30 min
3000     2 hrs 30 min
330   15 min
I have been using NL strips 3-10 pH
My sample is sperm membrne protein of dog, extracted in surfactant solution of Biorad.

[mdfenko]

are you sure you focus to completion?

do you fix the protein in the ief strip?

do you incubate the ief strip in sds sample buffer prior to running the second dimension?

[RanjnaSCheema]

mdfenko, on 29 February 2012 - 12:58 PM, said:

are you sure you focus to completion?

do you fix the protein in the ief strip?

do you incubate the ief strip in sds sample buffer prior to running the second dimension?

Yes, I am sure about focussing to completion and voltage reaches to 3000 volts as per the company's instructions.
Yes, i use IEF strips
Yes, I incubate IEF strip in Rehydration buffer containing SDS for minimum 16 hrs.

[mdfenko]

RanjnaSCheema, on 01 March 2012 - 09:48 AM, said:

mdfenko, on 29 February 2012 - 12:58 PM, said:

do you fix the protein in the ief strip?

Yes, i use IEF strips

is the protein fixed?

if so, how do you fix? how do you neutralize the fixative prior to incubating with sample buffer?

although 16 hours should be long enough, the protein may not have completely resolubilized in the sample buffer (does the buffer contain reducing agent as well as sds?).

[RanjnaSCheema]

mdfenko, on 01 March 2012 - 12:10 PM, said:

RanjnaSCheema, on 01 March 2012 - 09:48 AM, said:

mdfenko, on 29 February 2012 - 12:58 PM, said:

do you fix the protein in the ief strip?

Yes, i use IEF strips

is the protein fixed?

if so, how do you fix? how do you neutralize the fixative prior to incubating with sample buffer?

although 16 hours should be long enough, the protein may not have completely resolubilized in the sample buffer (does the buffer contain reducing agent as well as sds?).

I use following procedure for 2-D

1. I extract the sperm membrane proteins with readymade Biorad sol (5M urea, 2M Thiourea, 2% CHAPS, 1% DTT,, 2mM TBP,2% SB-3-10, 1 % carrier ampholytes, 40 mM Tris),
2. Rehydrate IEF strip (7 cm, pH 3-10) with 125 ul of above extract (100 ug protein) + 10 ul of test mixture, pH 3-10 for 16 hrs.
3. Then run the strip as per the protocol of company's instructions i.e.
Volts        Time
150     30 min
300     30 min
600     30 min
1500   30 min
3000   2 hrs 30 min
  330   15 min
4. Then equilibrate the strip in EB-1containing SDS and DTT and EB-2 containing SDS and Iodoacetamide for 10 min each. Wash in DW and then
     run in second dimension on 10% SDS-PAGE.

I want to clear one thing whether indicator dye completely disappears from tme strip after focussing.

[mdfenko]

indicator dye is not used in ief. it will not migrate the same way as in page (sds or native). it could change color due to pH differences.