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change of sample color to yellow after addind Sample diluting bufer


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#1 pakbiochemist

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Posted 16 October 2011 - 11:44 AM

Hi friends,
i have coem across a very strange situation today. i have been doing SDS PAGE since past 5yrs but this situation never happens. I extracted my histone proteins according to Sandra Hake Protocol (2007). and when added SDB (containg tris, mercaptoethanol, BPB, SDS and Glycerol) my sample turned yellow instead of blue and the colour doesnt change even after heating.

Please guide what might be the reasons of this color change

Regards

Binsan
PhD Student

#2 casandra

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Posted 16 October 2011 - 02:04 PM

Hi friends,
i have coem across a very strange situation today. i have been doing SDS PAGE since past 5yrs but this situation never happens. I extracted my histone proteins according to Sandra Hake Protocol (2007). and when added SDB (containg tris, mercaptoethanol, BPB, SDS and Glycerol) my sample turned yellow instead of blue and the colour doesnt change even after heating.

Please guide what might be the reasons of this color change

Regards

Binsan
PhD Student

change in pH........did you do an acid extraction (sorry am not familiar with the Hake protocol)?

Edited by casandra, 16 October 2011 - 02:05 PM.

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#3 protolder

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Posted 16 October 2011 - 10:08 PM

Hola, Casandra has reason the sample is too acid, add 2ul of (1 - 1.5 M ) tris 8.8 each time untill the colour turns to green and after to blue. Buena suerte

#4 pakbiochemist

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Posted 17 October 2011 - 05:52 AM

oh yes i have done acid extraction with 0.4N sulphuric acid and then precipitation with TCA.




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