10 replies to this topic

[Kami23]

Hey all,

I have been trying to make my reducing/non-reducing buffers for my protein sample and have been having a little problem with my dye. Each time i try to make it it turns out a different colour! I have had red and brown and yellow but never blue... does this matter for my western? this is the first time doing it on my own so i would like a little advice please

Thanks in advance

Kami

[dpo]

bromophenol's blue color is pH dependent:

Bromophenol blue is also used as a dye. At neutral pH, the dye absorbs red light most strongly and transmits blue light. Solutions of the dye therefore are blue. At low pH, the dye absorbs ultraviolet and blue light most strongly and appears yellow in solution. In solution at pH 3.6 (in the middle of the transition range of this pH indicator) obtained by dissolution in water without any pH adjustment, bromophenol blue has a characteristic green red color.

(http://www.answers.c...mophenol-blue-1)

so the color will depend on the pH of your buffer. If your buffer should have a neutral pH, but the bromophenol blue isn't blue, this means you have a problem with your pH

Edited by dpo, 28 October 2009 - 07:14 AM.

[Kami23]

dpo, on Oct 28 2009, 01:01 PM, said:

bromophenol blue's color is pH dependent:

Bromophenol blue is also used as a dye. At neutral pH, the dye absorbs red light most strongly and transmits blue light. Solutions of the dye therefore are blue. At low pH, the dye absorbs ultraviolet and blue light most strongly and appears yellow in solution. In solution at pH 3.6 (in the middle of the transition range of this pH indicator) obtained by dissolution in water without any pH adjustment, bromophenol blue has a characteristic green red color.

(http://www.answers.c...mophenol-blue-1)

so the color will depend on the pH of your buffer. If your buffer should have a neutral pH, but the bromophenol blue isn't blue, this means you have a problem with your pH

looking at it it looks like i havent made my Tris-HCl properly.. this could be why its yellow! If the buffer is too acidic will this affect the mobility of my protein in the gel?

[dpo]

I'm no protein gel specialist, but in a discontinous gel (with running and stacking gel), it's the pH which is different between the gels, so I could understand if an acidic sample will not run in the same way as a neutral sample. If you use the same Tris HCl buffer to make your gel, I think you will run into problems, but if it's only the sample, I have no idea whether the buffer in the gel can neutralize it enough ... If it's a precious sample, I would remake my Tris buffer, otherwise you can just give it a try and see what happens

[mdfenko]

Kami23, on Oct 28 2009, 09:19 AM, said:

looking at it it looks like i havent made my Tris-HCl properly.. this could be why its yellow! If the buffer is too acidic will this affect the mobility of my protein in the gel?

yes, (should run slower, if at all) at least until the electrode buffer overruns the sample (extreme stacking). if all your samples are in the same buffer then the effect may be uniform.

will be best to adjust the pH of the buffer.

[Kami23]

mdfenko, on Oct 28 2009, 02:23 PM, said:

Kami23, on Oct 28 2009, 09:19 AM, said:

looking at it it looks like i havent made my Tris-HCl properly.. this could be why its yellow! If the buffer is too acidic will this affect the mobility of my protein in the gel?

yes, (should run slower, if at all) at least until the electrode buffer overruns the sample (extreme stacking). if all your samples are in the same buffer then the effect may be uniform.

will be best to adjust the pH of the buffer.

right so i have to get the buffer right
Ok I added everything in a different order to the bromophenol blue and realised its the SDS thats making it go yellow... could it be out of date? (there isnt one on the bottle)... its a liquid one so could this be the problem?

[mdfenko]

the sds should not be acidic. low pH will cause the sds to decompose more rapidly.

do not use the acidic sds.

prepare fresh sds or purchase more.

(an old lot of powder may also cause trouble with electrophoresis).

[Kami23]

mdfenko, on Oct 28 2009, 06:28 PM, said:

the sds should not be acidic. low pH will cause the sds to decompose more rapidly.

do not use the acidic sds.

prepare fresh sds or purchase more.

(an old lot of powder may also cause trouble with electrophoresis).

Yep it was the SDS! I have fresh and my buffer is now blue... on closer inspection the old stuff was lumpy :S

Ok so now i have it how do i store it? ive seen all kinds of things like store in -20 in aliquots and others in fridge for no longer than a month etc....

Edited by Kami23, 29 October 2009 - 03:53 AM.

[mdfenko]

we never store sds in the refrigerator. it will crystallize at low temperature.

just make up enough for about a month and store on the bench (or in a cabinet).

[Kami23]

mdfenko, on Oct 29 2009, 02:46 PM, said:

we never store sds in the refrigerator. it will crystallize at low temperature.

just make up enough for about a month and store on the bench (or in a cabinet).

oops sorry Mdfenko i meant the reducing and non-reducing buffer i made

[mdfenko]

it also applies to the sample buffer. we store on the bench. you can freeze it (we freeze sample buffer with dtt), as well. i wouldn't store at 4C.