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What should be the pH of the leamlli sample buffer? - (Jan/22/2007 )

Hello all;

I'm making leamlli sample buffer to load my protein samples.
This is the recipie I'm using: 4x
1.5gTris
2.5g SDS
2.5mg Bromophenol Blue
20mL glycerol

complete to 50mL with dH2O

Now, everywhere I read, use TrisHCL solution at pH6.8
Should I just pH my whole solution to 6.8 since my tris is in powder??

Thanks

-medchemgirl-

QUOTE (medchemgirl @ Jan 22 2007, 02:32 PM)
Hello all;

I'm making leamlli sample buffer to load my protein samples.
This is the recipie I'm using: 4x
1.5gTris
2.5g SDS
2.5mg Bromophenol Blue
20mL glycerol

complete to 50mL with dH2O

Now, everywhere I read, use TrisHCL solution at pH6.8
Should I just pH my whole solution to 6.8 since my tris is in powder??

Thanks


No, you should make a stock solution of 1M or 2M Tris-HCl at pH6.8.

Hope this help.

-Minnie Mouse-

Really??? But I just made it like that and adjusted my pH to 6.8, so this is wrong??
wacko.gif


sad.gif

Will I have to remake it?

QUOTE (Minnie Mouse @ Jan 22 2007, 06:37 PM)
QUOTE (medchemgirl @ Jan 22 2007, 02:32 PM)
Hello all;

I'm making leamlli sample buffer to load my protein samples.
This is the recipie I'm using: 4x
1.5gTris
2.5g SDS
2.5mg Bromophenol Blue
20mL glycerol

complete to 50mL with dH2O

Now, everywhere I read, use TrisHCL solution at pH6.8
Should I just pH my whole solution to 6.8 since my tris is in powder??

Thanks


No, you should make a stock solution of 1M or 2M Tris-HCl at pH6.8.

Hope this help.

-medchemgirl-

I know someone did pH after making up the sample buffer, and the color of the buffer was brown instead of blue...may due to overshoot of pH

Just curious...
what color is your sample buffer?

Normally, people pH the Tris-HCl before making up the buffer, because it is very messy to pH sample buffer containing bromophenol blue....blue everywhere!!

Hope this help.

-Minnie Mouse-

QUOTE (Minnie Mouse @ Jan 23 2007, 11:12 AM)
I know someone did pH after making up the sample buffer, and the color of the buffer was brown instead of blue...may due to overshoot of pH

Just curious...
what color is your sample buffer?

Normally, people pH the Tris-HCl before making up the buffer, because it is very messy to pH sample buffer containing bromophenol blue....blue everywhere!!

Hope this help.


It might be best to make it up again as it's not hard to make, and is one of those things that is too fidgety to get wrong. If you make the stock Tris-HCl solution as suggested, it will always be there for other uses.

William John.

-WillCJohn-

Its true, it was a lit messy, but the color of my sample is still blue.



QUOTE (Minnie Mouse @ Jan 22 2007, 07:12 PM)
I know someone did pH after making up the sample buffer, and the color of the buffer was brown instead of blue...may due to overshoot of pH

Just curious...
what color is your sample buffer?

Normally, people pH the Tris-HCl before making up the buffer, because it is very messy to pH sample buffer containing bromophenol blue....blue everywhere!!

Hope this help.

-medchemgirl-

sds interferes with the functioning of the pH electrode. it crystallizes on the porous junction (because of the kcl). so, no, you should not adjust the pH of the sample solution after it is all together.

-mdfenko-

Ok, thanks a lot everyone, I just did a new buffer.
blush.gif

QUOTE (mdfenko @ Jan 23 2007, 03:44 PM)
sds interferes with the functioning of the pH electrode. it crystallizes on the porous junction (because of the kcl). so, no, you should not adjust the pH of the sample solution after it is all together.

-medchemgirl-