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western blotting - (Nov/28/2007 )

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hi,

i want to know the best time to transfer high molecular weight protein and also at what amphere?

-nadia naeem-

what is the size of the protein?

-scolix-

are you blotting wet or semi-dry?

sds-page?

-mdfenko-

QUOTE (scolix @ Nov 28 2007, 08:21 PM)
what is the size of the protein?


size of protein is 130 k dalton

-nadia naeem-

QUOTE (mdfenko @ Nov 28 2007, 08:23 PM)
are you blotting wet or semi-dry?

sds-page?


my blotting is transfering of bands from sds-page to nitrocellulose

-nadia naeem-

QUOTE (nadia naeem @ Nov 29 2007, 05:34 AM)
QUOTE (mdfenko @ Nov 28 2007, 08:23 PM)
are you blotting wet or semi-dry?

sds-page?


my blotting is transfering of bands from sds-page to nitrocellulose

is it a wet or semi-dry transfer?

do you add a little sds (0.05%) to your transfer buffer?

-mdfenko-

QUOTE (mdfenko @ Nov 29 2007, 11:38 PM)
QUOTE (nadia naeem @ Nov 29 2007, 05:34 AM)
QUOTE (mdfenko @ Nov 28 2007, 08:23 PM)
are you blotting wet or semi-dry?

sds-page?


my blotting is transfering of bands from sds-page to nitrocellulose

is it a wet or semi-dry transfer?

do you add a little sds (0.05%) to your transfer buffer?


no i have not add SDS in my transfer buffer

-nadia naeem-

QUOTE (nadia naeem @ Nov 30 2007, 12:21 AM)
QUOTE (mdfenko @ Nov 29 2007, 11:38 PM)
QUOTE (nadia naeem @ Nov 29 2007, 05:34 AM)
QUOTE (mdfenko @ Nov 28 2007, 08:23 PM)
are you blotting wet or semi-dry?

sds-page?


my blotting is transfering of bands from sds-page to nitrocellulose

is it a wet or semi-dry transfer?

do you add a little sds (0.05%) to your transfer buffer?


no i have not add SDS in my transfer buffer

addition of a little sds in the transfer buffer aids in the transfer of larger proteins.

without knowing the type of transfer you are performing it is hard to figure a good transfer time.

-mdfenko-

QUOTE
without knowing the type of transfer you are performing it is hard to figure a good transfer time.


I wouldn't mind a good reply where you gave different samples of transfer time/voltage and the rationale since I am (obviously) not doing the same WB as this person, but would love to understand the transfer better. Like the SDS thing is good to know (I'm assuming it keeps the protein from aggregating or something), for possible future studies.

Thanks

-MKR-

QUOTE (MKR @ Nov 30 2007, 01:20 PM)
QUOTE
without knowing the type of transfer you are performing it is hard to figure a good transfer time.


I wouldn't mind a good reply where you gave different samples of transfer time/voltage and the rationale since I am (obviously) not doing the same WB as this person, but would love to understand the transfer better. Like the SDS thing is good to know (I'm assuming it keeps the protein from aggregating or something), for possible future studies.

Thanks


do tank blotting O.N. (200 V/150 mA) with appropriate cooling; we use 20% methanol and 0.037 % SDS

-The Bearer-

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