black dot blackground on western - (Jan/19/2007 )
Hi together,
Its the first time that I´m here.
I often have dirty black dots on my film after development with ECL. I´m using 5% skim milk in TBST (0,2%) for blocking over night at 4°C and also for incubation of 1. antibody over night at 4°C. After 1 ab incubation I wash 3x 10min with TBST and the secondary AB I incubate for 1h at room temperature. After 3x 10 min wash I develop with ECL.
has anyone an idea of how I can reduce this unspecific background of distinct small black dots?
Thanks a lot !!
Bernd
Its the first time that I´m here.
I often have dirty black dots on my film after development with ECL. I´m using 5% skim milk in TBST (0,2%) for blocking over night at 4°C and also for incubation of 1. antibody over night at 4°C. After 1 ab incubation I wash 3x 10min with TBST and the secondary AB I incubate for 1h at room temperature. After 3x 10 min wash I develop with ECL.
has anyone an idea of how I can reduce this unspecific background of distinct small black dots?
Thanks a lot !!
Bernd
it looks like as has sth precipitated on your blots; is the skim milk filtered? check all other incubation solutions for precipitations especially the cooled solutions; also, Ab´s can precipitate if too old or wrong buffered
It seems strange, but it could be the powder on your gloves.
Try to get rid of the powder of your gloves (wash and dry with kimwipe) before to touch the ECL film.
It might be your secondary antibody. Try spinning it down for a couple of minutes.
your exposure cassette may be contaminated.
Its the first time that I´m here.
I often have dirty black dots on my film after development with ECL. I´m using 5% skim milk in TBST (0,2%) for blocking over night at 4°C and also for incubation of 1. antibody over night at 4°C. After 1 ab incubation I wash 3x 10min with TBST and the secondary AB I incubate for 1h at room temperature. After 3x 10 min wash I develop with ECL.
has anyone an idea of how I can reduce this unspecific background of distinct small black dots?
Thanks a lot !!
Bernd
Hi,
For the blocage stage, you can use 5% non fat milk in TBS/Tween during 2 hours max at RT!! Then, I incubate (without wash) the primary anticorps overnight at 4 °C, et then I wash the nitrocellulose membrane 3x15 min and 3 X 5min (total 1 hour), and I don't have Black backgroud!!!
Bye and good luck
two other things that may be happening:
rusty tweezers - leaves spots on the membrane
old detection reagent - leaves little clumpy spots as well
can you post a pic of your film?
use a large volume of wash buffer just in case some crud is sticking to your blot - and make sure you are shaking....
def agree with mdfenko -put a film in your empty cassette to make sure it isnt contaminated