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WB reference - Good reference for WB (Jul/29/2009 )

Does anyone a good and concise reference for WB? From preparation of stacking and running gel to the final result. I've read a very good one, from abcam (http://www.abcam.com/index.html?pageconfig=resource&rid=11375), but still I feel that there are some information missing.
It's because I've been working with different kinds of proteins, with a wide size range (i.e. 15kD - 370kD), un- and phosphorylated states,...
So, any help will be very welcome.

-Doda-

Doda on Jul 29 2009, 10:44 PM said:

Does anyone a good and concise reference for WB? From preparation of stacking and running gel to the final result. I've read a very good one, from abcam (http://www.abcam.com/index.html?pageconfig=resource&rid=11375), but still I feel that there are some information missing.
It's because I've been working with different kinds of proteins, with a wide size range (i.e. 15kD - 370kD), un- and phosphorylated states,...
So, any help will be very welcome.



Most protocols are the same with slight variation. It looks like you may be using lysates. If so, you have a wide variation of protein sizes that will transfer. They should all transfer well if you give them enough time and choose the proper membrane. I transferred very large proteins by doing them overnight at 30V (250mA and 90W) in the cold room and using a prestained marker to gauge it by. This is not a definate way to see if you have complete transfer, but it is as good as any.

To do this correctly, make sure that you are using PVDF as some of the smaller proteins can actually "blow through" nitrocellulose. PVDF is better for covalency. If you must use NC, put an additional sheet behind the first.

This is my protocol....Transfer overnight. Block for 1 hour using 5% milkblot (in TBS-T20 1% or PBS-T20 1%). Remove block and immediately add your primary (1:2000 to 1:10,000 dilution depending on sensitivity) diluted in 5% milkblot. Incubate shaking for 1 hour. Wash with your TBS-T or PBS-T for 3 washes of 15 minutes each. Incubate with your secondary in 5% milkblot dilution at 1:5000 or 1:10,000 depending on sensitivity for 30 minutes. Repeat washes as above. The secondary should have a conjugation of HRP or alkaline phosphatase. Use which ever to image your blot.

I have done hundreds of western blots using this protocol. Some I've had to use another block besides milkblot. If you do, you can use BSA or just increase your tween-20 concentration.

Best of Luck!

-BIOKMST-

google Laemmli

-bob1-

Thanks a lot BIOKMST; I'll definitely try your protocol. Oh, and about the running time, how long do you leave it? I was running at 120V for 90min and it looked like the protein kinda got stuck at the stack-separating gel interphase. The protein is 350kD and I run it into 6% gel. Any idea?
Anyways, I'll try transferring overnight at low voltage. Thanks!



BIOKMST on Jul 31 2009, 12:03 AM said:

Doda on Jul 29 2009, 10:44 PM said:

Does anyone a good and concise reference for WB? From preparation of stacking and running gel to the final result. I've read a very good one, from abcam (http://www.abcam.com/index.html?pageconfig=resource&rid=11375), but still I feel that there are some information missing.
It's because I've been working with different kinds of proteins, with a wide size range (i.e. 15kD - 370kD), un- and phosphorylated states,...
So, any help will be very welcome.



Most protocols are the same with slight variation. It looks like you may be using lysates. If so, you have a wide variation of protein sizes that will transfer. They should all transfer well if you give them enough time and choose the proper membrane. I transferred very large proteins by doing them overnight at 30V (250mA and 90W) in the cold room and using a prestained marker to gauge it by. This is not a definate way to see if you have complete transfer, but it is as good as any.

To do this correctly, make sure that you are using PVDF as some of the smaller proteins can actually "blow through" nitrocellulose. PVDF is better for covalency. If you must use NC, put an additional sheet behind the first.

This is my protocol....Transfer overnight. Block for 1 hour using 5% milkblot (in TBS-T20 1% or PBS-T20 1%). Remove block and immediately add your primary (1:2000 to 1:10,000 dilution depending on sensitivity) diluted in 5% milkblot. Incubate shaking for 1 hour. Wash with your TBS-T or PBS-T for 3 washes of 15 minutes each. Incubate with your secondary in 5% milkblot dilution at 1:5000 or 1:10,000 depending on sensitivity for 30 minutes. Repeat washes as above. The secondary should have a conjugation of HRP or alkaline phosphatase. Use which ever to image your blot.

I have done hundreds of western blots using this protocol. Some I've had to use another block besides milkblot. If you do, you can use BSA or just increase your tween-20 concentration.

Best of Luck!

-Doda-

What about it, bob1? That's the sample buffer I use for my samples.

bob1 on Jul 31 2009, 09:19 AM said:

google Laemmli

-Doda-

Laemmli wrote the original denaturing SDS-PAGE protocols and papers, that's why the buffer is named after him!

-bob1-

bob1 on Aug 3 2009, 09:24 AM said:

Laemmli wrote the original denaturing SDS-PAGE protocols and papers, that's why the buffer is named after him!


oh... ok, thanks!

-Doda-