[Hamid]

Hi Shanshan,

With regards to you seeing multiple bands on your westerns, it is quite normal. That is occurring because most antibodies for western, with the exception of a few histone ones, are not monoclonal, so there were be background bands. Even quite a few monoclonal antibodies will recognize more than one epitope.
Also when working with tissues, you will have higher background due to the presence of antibodies against the animal used to generate the antibodies.
When we run westerns for epitope integrity, we cut the membrane such that it incorporates the area representing the size of the protein of interest. By doing that you reduce the amount of antibody you are using, but also render the binding to the region of interest more efficient.

Can you post your post IP bioanalyzer results as well as your western if possible?
Did you run the post IP DNA on a high sensitivity chip? If so, then the high sensitivity chip is very sensitive to salts, and will always give a higher size range and distribution as compared to the regular sensitivity chip. We are almost done with a technical note that clearly shows that and should have that available for posting by next week.


Thank you

hamid

[shanshanzhou]

Oh, I also tried to filter out the "crap" using miracloth and shear after cleaning. Got the same result with crude lysate other than reduced amount of DNA recovered...

Shanshan

[shanshanzhou]

Good thoughts, wasted so much antibody on westerns

[shanshanzhou]

Hi Shanshan,

With regards to you seeing multiple bands on your westerns, it is quite normal. That is occurring because most antibodies for western, with the exception of a few histone ones, are not monoclonal, so there were be background bands. Even quite a few monoclonal antibodies will recognize more than one epitope.
Also when working with tissues, you will have higher background due to the presence of antibodies against the animal used to generate the antibodies.
When we run westerns for epitope integrity, we cut the membrane such that it incorporates the area representing the size of the protein of interest. By doing that you reduce the amount of antibody you are using, but also render the binding to the region of interest more efficient.

Can you post your post IP bioanalyzer results as well as your western if possible?
Did you run the post IP DNA on a high sensitivity chip? If so, then the high sensitivity chip is very sensitive to salts, and will always give a higher size range and distribution as compared to the regular sensitivity chip. We are almost done with a technical note that clearly shows that and should have that available for posting by next week.


Thank you

hamid

Yep, I used the high sensitivity chip, and I used EB buffer come with the column from Qiagen. Please see attachment for the post IP results, I'll try to take a pic of my western result later.

I attached another older pre-CHIP bioanalyzer results from several samples with time courses (sorry it's not labeled), even for the 'good' ones, the peak doesn't drop very fast dragging a long tail towards bigger fragment. Is this worrisome to you? Or its just different properties from different cell types.
Thanks so much!

Best,
Shanshan

[tamara.tjitrowirjo]

Hi...

I have ran Western blot with my ChIP and Input (only sheared chromatin without any IP) samples. I did not run reverse cross-linking. It depends on the condition of your Western blot. We use sample buffer containing beta-mercaptoethanol and boil them for 5 minutes. This will denature the protein and release all the DNA that are bound, essentially the same as reverse cross-linking procedure. I then ran them on a denaturing gel and probe with antibody against my protein of interest. It works well.

I sheared my samples using Branson 250D probe sonicator for 15 cycles (total ON sonication is 7 minutes 30 seconds).

[pmiguel]

Boiling DNA that you want to sequence seems crazy to me. But maybe the BME prevents some of the damage that would otherwise occur?

--
Phillip

[tamara.tjitrowirjo]

Noooooo Phillip... This is for running the Western. I would never boil my DNA that will be sequenced... Those DNA for sequencing undergoes reverse cross-linking at 65C overnight followed by RNase A and Proteinase K treatment and then phenol chloroform isolation and then sample prep.

[pmiguel]

Ah. Thanks for the clarification.

--
Phillip

[shanshanzhou]

Hi...

I have ran Western blot with my ChIP and Input (only sheared chromatin without any IP) samples. I did not run reverse cross-linking. It depends on the condition of your Western blot. We use sample buffer containing beta-mercaptoethanol and boil them for 5 minutes. This will denature the protein and release all the DNA that are bound, essentially the same as reverse cross-linking procedure. I then ran them on a denaturing gel and probe with antibody against my protein of interest. It works well.

I sheared my samples using Branson 250D probe sonicator for 15 cycles (total ON sonication is 7 minutes 30 seconds).

That's a nice band, I did the same by boiling 5 min and got really messy stain, start thinking maybe its the antibody I use. Trying again with some reverse crosslinked sample today, see if there will be a difference. What power setting do you use on the Branson? We have a digital fisher one that allow you to change Joule and %Amplitude, I can understand %Amplitude, don't know what should I set for Joule...

Shanshan

[tamara.tjitrowirjo]

hi Shanshan,

Thanks for the comment! =) I am very happy with the Western...

You need to optimize it based on your cells. But from our experience since they are all nuclei extracted, they differ only slightly. But then again, all our cells are from human breast.

Amplitude setting for 50%, we do not have Joule setting so I don't know anything about it. Good luck!

[shanshanzhou]

hi Shanshan,

Thanks for the comment! =) I am very happy with the Western...

You need to optimize it based on your cells. But from our experience since they are all nuclei extracted, they differ only slightly. But then again, all our cells are from human breast.

Amplitude setting for 50%, we do not have Joule setting so I don't know anything about it. Good luck!

Thanks tamara

[ALive]

Hi Everyone,

I've done some Western blotting, and I didn't see a decrease in the amount of protein as the sonication time was increased... The conditions I tried were:

2 minutes cross-linking -> sonication for 5 minutes; 15 minutes; 20 minutes
5 minutes cross-linking -> sonication for 5 minutes; 15 minutes; 20 minutes
8 minutes cross-linking -> sonication for 5 minutes; 15 minutes; 20 minutes

To prepare the samples, I used sample buffer containing beta-mercaptoethanol and put them >90C for 10 minutes. But I was seeing two bands - the smaller was the correct size, and the larger looked almost double the size so could be a dimer (? this is my first time Western blotting, I don't know a lot about them ). So, I reverse cross-linked the samples and then did the same sample buffer step as above. This seemed to almost get rid of the larger band.

I've attached a picture of a western I did today.

1: Input - sample buffer
2. Input - reverse cross-linked + sample buffer
3. 2 minute cross-link, 5 minute sonication - sample buffer
4. 2 minute cross-link, 20 minute sonication - sample buffer
5. 2 minute cross-link, 20 minute sonication - reverse cross-linked + sample buffer
6. 8 minute cross-link, 5 minute sonication - sample buffer
7. 8 minute cross-link, 20 minute sonication - sample buffer
8. 8 minute cross-link, 20 minute sonication - reverse cross-linked + sample buffer

So it looks to me like I'm not destroying the epitope by over-sonicating... Maybe I’m not using enough protease inhibitors in the antibody incubation step or wash step?

Thanks in advance for any comments

Ali

[Hamid]

Hi Alive,

The western does look good, and your protein is quiet small in size which should render it more resistant to damage during shearing. It does seem like it is since I don't see a major difference between the 5 minute, and 20 minute processing. Your protein seems quiet abundant . One thing to remember is that just because you are seeing the band on the western, it does not not mean that it is still associated or linked to the DNA fragment. The western will still pickup proteins not associated with the chromatin. The only way to find that out will be do do an IP.
My suggestion is that you pick the samples that generated the best shearing profile based on the bioanalyzer results, and do an IP on a 5 minute and 20 minutes processed samples. comparing the post-IP bioanalyzer will then give you a good indication of your epitope(protein+chromatin) integrity.

Thank you

Hamid

[ALive]

Thanks Hamid. I'll do that and post my results.

Btw, I'm looking at histone modifications and used anti H4K20me1 for the Western.

Cheers, Ali

[tamara.tjitrowirjo]

Hello people...!!

Forgot to report back. I had that problem where I extracted my ChIP sample prep DNA on gel about 200-300bp (with adapters) and run bioanalyzer and caliper and both of them reported that my fragments are about 4-5kb! I went on to sequence them anyways and they are about 150bp (without adapters). Got sequencing results okay, no problem with extra large fragments.

Hard evidence that I cannot trust the bioanalyzer and caliper, at least for my case. Good old gel is trustworthy! =D