blotting detection - (Jul/26/2011 )

Hi,

Its first time I am doing western blot independantly, After transfer into PVDF membrane, I blocked the membrane in BSA overnight and then incubate with primary Ab overnight and after wash incubate 2hrs in secondary Ab. When time came for detection I came to know that Film developer is out of work in our department. I am wondering can I keep the membrane may be in TPBS untill Film developer back to work? Or is there any other method to detect HRP conjugated secondary Ab? Any suggestion will be appreciated

thanks

Sagaguchi on Tue Jul 26 18:50:13 2011 said:

yeah you can keep the membrane at 4 degree as casandra suggested, and i too feel your blocking time is too long, though i can somehow agree with your probing time with primary... Anyhow fine if that is your optimized western...then regarding deveoping, you can even do it manually, may be you will have the developer and fixer solution in your developing room, pour them in two seperate small trays and dip your exposed film first in develper and then into fixer in the dark room and finally wash in water...i have done the same when ours was out of order.. give a try.....

casandra on Tue Jul 26 19:55:03 2011 said:

Sagaguchi on Tue Jul 26 18:50:13 2011 said:

Hi,

Its first time I am doing western blot independantly, After transfer into PVDF membrane, I blocked the membrane in BSA overnight and then incubate with primary Ab overnight and after wash incubate 2hrs in secondary Ab. When time came for detection I came to know that Film developer is out of work in our department. I am wondering can I keep the membrane may be in TPBS untill Film developer back to work? Or is there any other method to detect HRP conjugated secondary Ab? Any suggestion will be appreciated

thanks

hi Sagaguchi...yup, you can keep your membranes in PBST in the fridge meanwhile but if you really need the results...couldn't you find a film developer in another department or a CCD camera imager? Btw, is your western blotting protocol optimised......you do 2 O/N incubations?....that's a looong.....

Thanks everyone, I have tried to develope after a week and it worked well.

GNANA on Wed Jul 27 07:03:54 2011 said:

yeah you can keep the membrane at 4 degree as casandra suggested, and i too feel your blocking time is too long, though i can somehow agree with your probing time with primary... Anyhow fine if that is your optimized western...then regarding deveoping, you can even do it manually, may be you will have the developer and fixer solution in your developing room, pour them in two seperate small trays and dip your exposed film first in develper and then into fixer in the dark room and finally wash in water...i have done the same when ours was out of order.. give a try.....

casandra on Tue Jul 26 19:55:03 2011 said:

Sagaguchi on Tue Jul 26 18:50:13 2011 said:

Hi,

Its first time I am doing western blot independantly, After transfer into PVDF membrane, I blocked the membrane in BSA overnight and then incubate with primary Ab overnight and after wash incubate 2hrs in secondary Ab. When time came for detection I came to know that Film developer is out of work in our department. I am wondering can I keep the membrane may be in TPBS untill Film developer back to work? Or is there any other method to detect HRP conjugated secondary Ab? Any suggestion will be appreciated

thanks

hi Sagaguchi...yup, you can keep your membranes in PBST in the fridge meanwhile but if you really need the results...couldn't you find a film developer in another department or a CCD camera imager? Btw, is your western blotting protocol optimised......you do 2 O/N incubations?....that's a looong.....