5 replies to this topic

[rsphorler]

Hello

I started to do some work with Gel shifts and am getting results where the free probe runs at a certain point on the gel and when i add whole cell extract the band move higher in the gel and higher still as the conc. of WCE added increases. I don't see a shift between a lower free probe form and a higher bound probe form that I would expect just this slower migration as [WCE] increases. The control which is WCE of a culture not expressing my protein of interest does not shift or move the band when the [WCE] increases.

Has anyone seen this before and does anyone know what i shappening to the probe?

Thanks

Richard

Edited by rsphorler, 09 April 2010 - 07:48 AM.

[swanny]

Do you have an image to post?
Does the probe shift in discrete bands, or is it a smear? I'm guessing it will be a smear, but I thought I'd just check.

[huotoad]

swanny, on May 5 2010, 05:50 PM, said:

Do you have an image to post?
Does the probe shift in discrete bands, or is it a smear? I'm guessing it will be a smear, but I thought I'd just check.

Hi Swanny,
I am trying to do EMSA recently. I extracted nuclear protein from lettuce seeds. I run a protein gel and all of people who have some protein experience told me the proteins are looking good. I got lots of protein bands. most of them are bigger than 65KD. and there two strong clusters containing about 5-8 bands in each cluster. protein below and above two clusters are weak.

I also tried several times of gel shift assay. I see the free probe but no binding. most of time, I have similar phenotypes to the one described by the person above. I am wondering if my protein is functional. I used TATABOX1 as postitive control to confirm if the protein has binding capacity, but I got a artifact band in the well without my nuclear extract. What postitive control did you use? The binding system from DIG gel shift kit works well for the control from the kit.

By the way, do u think that it is good to use long DNA probe, say 200-300bp? my probe is only 50bp, however, I really don't know if my probe has truly binding sites.

Your suggestion would be very welcome.

[swanny]

huotoad, on May 21 2010, 02:37 PM, said:

swanny, on May 5 2010, 05:50 PM, said:

Do you have an image to post?
Does the probe shift in discrete bands, or is it a smear? I'm guessing it will be a smear, but I thought I'd just check.

Hi Swanny,
I am trying to do EMSA recently. I extracted nuclear protein from lettuce seeds. I run a protein gel and all of people who have some protein experience told me the proteins are looking good. I got lots of protein bands. most of them are bigger than 65KD. and there two strong clusters containing about 5-8 bands in each cluster. protein below and above two clusters are weak.

I also tried several times of gel shift assay. I see the free probe but no binding. most of time, I have similar phenotypes to the one described by the person above. I am wondering if my protein is functional. I used TATABOX1 as postitive control to confirm if the protein has binding capacity, but I got a artifact band in the well without my nuclear extract. What postitive control did you use? The binding system from DIG gel shift kit works well for the control from the kit.

Quote

I didn't use a positive control. I used GST as a negative control, and to control for any GST traces in the prep.

Quote

By the way, do u think that it is good to use long DNA probe, say 200-300bp? my probe is only 50bp, however, I really don't know if my probe has truly binding sites.

Quote

My probes ranged from 90 bp down to only 15, and they all worked fine. You will always need to have decent binding kinetics to see anything. If you're confident you should have some binders, but aren't getting any on your gels, try changing the buffer conditions - salt and pH would be my suggestions for starting points. It's not unusual to have to finetune the binding conditions, but I'm no expert on that part (aboutthe only part of my PhD where I got lucky!!).

[huotoad]

swanny, on May 20 2010, 10:33 PM, said:

huotoad, on May 21 2010, 02:37 PM, said:

swanny, on May 5 2010, 05:50 PM, said:

Do you have an image to post?
Does the probe shift in discrete bands, or is it a smear? I'm guessing it will be a smear, but I thought I'd just check.

In the first try, the free probe is looking good, which means there are no smear or discrete bands. in my second and third tries, they are smears

Hi Swanny,
I am trying to do EMSA recently. I extracted nuclear protein from lettuce seeds. I run a protein gel and all of people who have some protein experience told me the proteins are looking good. I got lots of protein bands. most of them are bigger than 65KD. and there two strong clusters containing about 5-8 bands in each cluster. protein below and above two clusters are weak.

I also tried several times of gel shift assay. I see the free probe but no binding. most of time, I have similar phenotypes to the one described by the person above. I am wondering if my protein is functional. I used TATABOX1 as postitive control to confirm if the protein has binding capacity, but I got a artifact band in the well without my nuclear extract. What postitive control did you use? The binding system from DIG gel shift kit works well for the control from the kit.

Quote

I didn't use a positive control. I used GST as a negative control, and to control for any GST traces in the prep.

are you using purified specific protein? Mine is whole cells extract.

Quote

By the way, do u think that it is good to use long DNA probe, say 200-300bp? my probe is only 50bp, however, I really don't know if my probe has truly binding sites.

Quote

My probes ranged from 90 bp down to only 15, and they all worked fine. You will always need to have decent binding kinetics to see anything. If you're confident you should have some binders, but aren't getting any on your gels, try changing the buffer conditions - salt and pH would be my suggestions for starting points. It's not unusual to have to finetune the binding conditions, but I'm no expert on that part (aboutthe only part of my PhD where I got lucky!!).

[swanny]

Purified protein.