Hi Check you running buffer recipe ..SDS should be 1% in the running buffer.

Hi Khadar thanks for your response.

Here is my running buffer recipe I have been using:

Tris base 0.025 M, Glycine 0.192 M, SDS 0.1% (w/v) and the final pH of the buffer adjusted to 8.3.

So according to you increasing the SDS concentration in running buffer to 1% will help to reduce the vertical streaking.

usual recipe for 10X 1 L running buffer is

Tris-base - 30g , Glycine - 144g and SDS 10g.

You never adjust pH for electrophoresis. Just add the salts and add 900ml water and after the salts are dissolved make upto 1L. DO NOT adjust pH at all. You use it directly diluting to 1X.

Equilibration (Eq) step appears to be affected. I see that you have only one Eq Buffer. In the past I used two Eq buffers one consisting of DTT followed by another containing IDA. So total Eq time was 30' (15' each) This will give good amount of time for SDS to equilibriate. Also I made these Eq Buffer Fresh always.

I am really sorry but one should not use 1% SDS for running gel, Khadar's recipie also gives you 0.1% SDS to a final concenteration.

Yes I meant in making the running buffer we make 1% and when diluted it does goes to 0.1% . I did had this problem before so I can surely say this must be the problem.And also please check how much is the limitation of protein you can load on a 11cm strip. I used to use 400mg for 24cm strip.

Hi Khadar,

When I made the running buffer, I made directly 1X running buffer (Tris Base 6.057 g, Glycine 28.83 g and SDS 2g to adjust the final volume to 2 lit). So I have followed the same recipe like the one you suggested above. The only difference was I tried to adjust the pH to 8.3, I will not adjust the pH next time.

Do you think any other modification in the current protocol will help to reduce vertical streaking. For example, decreasing the protein load, increasing the equilibration time.

How would you rate the current protein gel image on a 1-10 scale with 1 being the worst and 10 being the best.

Thanks

Monil Desai.

Hi Deepak,

Thanks for your response

I also did two equilibration steps. For Eq 1, I added 2% SDS and for Eq 2, I added 2.5 % Iodoacetamide that were freshly weighed and added. I did both the steps 15 min each.

Do you suggest any other modification in the current protocol that will help to reduce the vertical streaking?

If you observe closely the vertical streaking is more pronounced in the higher molecular weight region and also at the tip of the protein spot.

How would you rate the current protein gel image on a 1-10 scale with 1 being the worst and 10 being the best.

Monil Desai

Hi Khadar,

Is there any particular reason that we first make 10X solution and then dilute to 1X solution to get the final concentration of SDS to 0.1%.

The recommended protein load for 11 cm IPG strip is 100- 200 microgram for Commassie blue staining and maximum protein loading is 1 mg for 11 cm IPG srtrip. I am loading approx 650 microgram.

Monil

So that is a good reason why I think the equilibration is a problem as you see the streaking is more prominent in high molecular weight proteins. I think increasing the incubation to about 40-50 min may help. (I have never done this myself though, as I did not encounter this problem of vertical streaking).

Alternatively as suggested you may decrease the amount of protein.

Out of curiosity, what exactly is this 2DE gel for, is it for western blotting or identification of differentially expressed proteins by MS. Is that a silver stained gel or a comassie?

I will increase the equilibration time to 30 min each and see if it helps to reduce the vertical streaking and also decrease the protein load.

The 2DE gel is for identification of differentially expressed proteins by MS and I have done Comassie blue staining.

vertical streaking happens because of isoelectrical precipitation of proteins, overfocusing can lead to this as well as high protein load, bad equilibration and protein refolding, etc.

this problems can be prevented by being sure that the first dimension is not conducted longer than necessary, if you have high protein load, you could prevent the streaking by prolonging the equilibration time or by lowering the protein load and adding a more sensitive stain.

more tips can be found here

http://download.bioon.com.cn/upload/month_1002/20100226_48b830949c7f8d38b95bDTuiAK2Oh7xE.attach.pdf

Hi Mauricio,

Thanks for your response. Currently, I am loading approx. 650 microgram protein which is relatively high for pH 3-10 11 cm gel and also focusing for approx 60,000 Vh. In the next trial, I plan to reduce the protein load to approx 350 microgram which will help me in three ways: proper equilibration (interaction of SDS with protein), proper protein clean up (using 2D protein clean up kit) and reduce vertical streaking. Also, I will reduce the focusing to 35,000 Vh. I will keep you posted with my next results.

Hi Everyone,

Thank you for your suggestions until now.

I am in process of running four gels (pH 3-10 11 cm with approx 325 micrograms protein laod, IEF focusing 36,000 Vh, 12% SDS PAGE gel). I will be uploading the gel images day after tomorrow. Please provide your feedback and suggestions for the gel images.

Thanks

Monil Desai.

Hi Everyone,

Please find attached the four gel images.

Most of the gel conditions remain the same except:

For all the four gels I loaded 325 ug protein load on pH 3-10, 11 cm IPG strip and focusing was done for 36,000 VH.

I am still observing some vertical streaking in higher molecular weight region.

Kindly suggest some changes to reduce the vertical streaking.

Thanks

Monil Desai.

So although a bit late in reply, but your gel looks much better in quality. I think your major reason was protein amount. hence you can further decrease your protein amount (may be 200 ug) and do a silver staining. this will at least help you identify if there are any differentially expressed proteins (and ofcourse a good picture for publication). Once you know there are differentially expressed proteins (based on sliver staining) then you may order the strips in the range of your POI and relevant amount of protein (you may keep about 200-250 ug for 11 cm strip) and do a colloidal comassie staining.