Difference between PBS/TBS and Tween/TritonX?

[trypsin]

Hello,
i would like to know if someone knows what the difference between TBS and PBS buffer is? some people use PBS and other TBS for the wasch-solutions (westernblot).

I got a blot where the signal was very strong (ECL) but i couldn't see anything on the picture (lumineszenzreader)- Why?

i will be happy if someone can answer.
trypsin

[keithbrent2000]

Hi,

TBS uses Tris as the buffer, and PBS uses phosphate as the buffer. They should give similar results. Most people use tween as the detergent for washing, and it is generally not as strong as triton. You may want to ask someone to show you if you are using the lumineszenzreader correctly.

[trypsin]

Hi,
thanks for your answer.
Is it possible that the phosphate (PBS) react with small molecules and change the result?

I bought a pure protein which was 8kDa big. I should get one or two bands. I've got two bands but there are more bands in the higher MW-range and the signal is very strong between the two bands. Under my two bands i could see white bands. And my blot has a background and the marker is stained too.

Isn't my secondar antibody specific? It was add in 5% low fat milk in TBS + 0,1% Tween.
Should i try to dilute the antibody more or change the buffer (without lowfatmilk or Tween)?

trypsin

[JINCAO]

Hi,
According to the Pierce's Western Handbook, you should choose PBS or TBS buffer according to your detection kit. If you use AP conjugated secondary antibody, you should use TBS, as PBS interefere with alkaline phosphatase.