It looks like you may have overloaded the sample in addition to over developing the silver. I would try loading half as much protein and quench the silver staining solution a little sooner

Xiaojing Sui,

For lysis ~ 230–260 mg wet weight of frozen (-70DC) hippocampal tissue you need

1.4 volumes of 9.6 M urea,

2. 4% of CHAPS,

3.70 mM dithiothreitol (DTT);

4.5% IPG buffer immobilized pH gradient buffer with pH 3–10.

(https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314807262343/litdoc71500582AH_20110831223225.pdf)

Homogenize the tissue in an Eppendorf tube onice with a sonicator in 3-6 pulses of 10 sec

Spin in ultracentrifuge for 1 hr x 100,000 g.

Collect supernatants.

*** The clean-up form the lipids you may carry out by sucrose cushion (gradient) or by 2D Clean-up Kit https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314729545976/litdoc80648660AE_20110830213806.pdf

Good luck

Hi Joshua, at first I intended to perform the Coomassie blue staining. However, since the result did not show as many spots as possible, I conducted silver-staining after coomassie blue staining. The trouble bothered me was the vertical and horizontal streak. I am not sure if it was due to the impurity of the sample.

Hi Anna,

Thanks for your help. My mentor told me that using 2D clean-up kit will reduce some proteins. I have looked up related literature for hippocampus protein extraction. Some of them used TCA-percipitation method. So I still want to try this method. However, some experimental details i can not make sure. I am still not sure if those steaks are due to the impurities of the proteins. Do you have similar results like that? Thank you.

HI xia,

I faced the same streak problem while working with C. elegans proteome, i tried 2D Clean up kit from GE and avoided the streaks. Better you try this.. Wish you gud luck

Hi Durai,

Thanks for your help. Do you also regard the streak as the result of impurity of proteins? Maybe I should try 2D-clean up kit. Thanks.

Hi Xiaojing Sui,

I have performed many 2Ds with protein extracts from rat hippocamus, but never have to use precipitation methods or any Clean up kit. Please take a look at my publications for the recipes i ve used and the amount of proteinloads per IPG/Strip. I would really omit clean up methods since they will add recovery issues to your 2D methodology.

good luck,

Murat

Hi Xiaojing,

It seems to me protein overload effects more than contamination effects, as other contributors said. You may try with clean up kits, we used them with good recovery efficiency (about 95%) and the gel resolution is better (but not so much...), however you can lose some low abundance proteins, so the decision is up to you... One thing, please be sure that you use reagents fresh prepared, especially with DTT for the first dimension electrophoresis!!!

Please find below some references that might be helpful:

Heppelmann et al. (2007), Electrophoresis, 28, 3988-3991

Görg et al. (2009), Electrophoresis, 30, S122-S132

Wu et al. (2009), Proteome Sicence, 7:38 (doi:10.1186/1477-5956-7-38) open access

Valente et al. (2012), Electrophoresis, 33, 1947–1957

Cheers,

Miguel

Hi Murat,

I have read your publications. So I want to make sure that your procedure is 1) homogenizing the brain tissue in lysis buffer, 2) then centrifuge at 20000 g for 60 min to remove the debris. My problem is that I do not have a homogenizer, can i use the sonication instead? Thank you.

Hi Miguel,

Thank you for your help. The coomassie blue staining result also showed the vertical streak and horizontal streak. But the spots were not too clear to distinguish. So I performed the silver staining. DTT is added freshly. Thank you for your advice.

Cheers,

Xiaojing

Hi Xiaojing,

These papers can help you get better gels.

Butt RH, Coorssen JR (2005) Postfractionation for enhanced proteomic analyses: routine electrophoretic methods increase the resolution of standard 2D-PAGE. J Proteome Res 4: 982-991.

Butt RH, Lee MW, Pirshahid SA et al. (2006) An initial proteomic analysis of human preterm labor: placental membranes. J Proteome Res 5: 3161-3172.

Butt RH, Coorssen JR (2006) Pre-extraction sample handling by automated frozen disruption significantly improves subsequent proteomic analyses. J Proteome Res 5: 437-448.

Tatiana

The new coomassie blue staining result.

Hi Xia,

Hope the gel is better now, You can still filter all your reagents to avoid the streaking effect

Wish you gud luck

@Durai,

Hi, do you think the vertical streak is due to the over-IEF focus? The IEF ends at 110 KVh. Thank you.

hi Xia,

the vertical streaks are due to low concentration of SDS in cathode buffer. Try to use higher concentration of SDS in the running buffer (Cathode bufffer)

Hi Nishad,

Thanks for your reply. I have used higher concentration of SDS in the running buffer and get the satisfying result.

Cheers,

Xiaojing

If you do not have a motorized tissue homognizer, you can use RIPA buffer, then clean up sample using G-Biosciences Perfect Focus. There are other reagents from G-Biosciences that will do an excellent job in extracting proteins from hippocampus.

Automated frozen disruption gives better results than homogenization. (Butt RH, Coorssen JR (2006) Pre-extraction sample handling by automated frozen disruption significantly improves subsequent proteomic analyses. J Proteome Res 5: 437-448.)

How we can extract proteins from gram positive bacteria eg. staph aureus? for 2D electrophoresis?

I think you have oriented your your Strips wrong in the second dimension , make sure to face the plastic side with the glass and the gel side facing you(in the front).and also I have used passive condition in the 1th dimension, it worked with me I do not know why you use active rehydration.

I suggest you to use Katrine Marcus methode.

May be your proteins are getting diffused during the equilibration step before the SDS-PAGE.