2 replies to this topic

[HLee]

Hi,

I'm doing IP with 2 purified protein to see their interaction.
One is tagged w/ GFP and the other is w/ myc.

I IP w/ aGFP ab (rabbit) then blotted w/ a-myc ab (mouse) or a-GFP ab (same one used at IP).

Blotting /w a-myc ab,
I can see very strong 2 bands at ~ 25 kDa and 53kDa.
25 kDa band is more than 5 times stronger than 52kDa
100% input of myc-tagged protein (~33 kDa) shows much weaker (<1/10) slgnal than those 2 bands.
100% input of GFP-tagged protein didn't show any signal.

Blotting w/ a-GFP ab,
IP shows strong 3 bands at ~ 25 kDa and 53kDa (here, IgG light and heavy chain, absolutely) and 28 kDa GFP-tagged protein
52 kDa bans is ~ 3 times stronger than 25 kDa

With their size, two bands blotted w/ a-myc seems like IgG. But it doesn't make sense at all.
How a-mouse secondary antibody detects antibody from rabbit.
And the patterns of signal strentgh of two bans are differen from blots w/ a-Myc and a-GFP ab.

But, I just mixed 2 PURIFIED protein for IP.
So, in the tube there's no proteins other than 2 proteins and a-GFP antibodies.

Anyone can think what's hapenning, what are those two bands, and ** how to remove those bands!!!!

(a-GFP ab is A11122, Invitrogen.
I used protein G-dynabeads.
I followed conventional protocol of IP excepting using cell lysates but mixing 2proteins.
During IP, all buffer is PBS w/ 1% Empigen (detergent))

Edited by HLee, 15 June 2009 - 12:34 PM.

[smu]

HLee, on Jun 15 2009, 12:21 PM, said:

Hi,

I'm doing IP with 2 purified protein to see their interaction.
One is tagged w/ GFP and the other is w/ myc.

I IP w/ aGFP ab (rabbit) then blotted w/ a-myc ab (mouse) or a-GFP ab (same one used at IP).

Blotting /w a-myc ab,
I can see very strong 2 bands at ~ 25 kDa and 53kDa.
25 kDa band is more than 5 times stronger than 52kDa
100% input of myc-tagged protein (~33 kDa) shows much weaker (<1/10) slgnal than those 2 bands.
100% input of GFP-tagged protein didn't show any signal.

Blotting w/ a-GFP ab,
IP shows strong 3 bands at ~ 25 kDa and 53kDa (here, IgG light and heavy chain, absolutely) and 28 kDa GFP-tagged protein
52 kDa bans is ~ 3 times stronger than 25 kDa

With their size, two bands blotted w/ a-myc seems like IgG. But it doesn't make sense at all.
How a-mouse secondary antibody detects antibody from rabbit.
And the patterns of signal strentgh of two bans are differen from blots w/ a-Myc and a-GFP ab.

But, I just mixed 2 PURIFIED protein for IP.
So, in the tube there's no proteins other than 2 proteins and a-GFP antibodies.

Anyone can think what's hapenning, what are those two bands, and ** how to remove those bands!!!!

(a-GFP ab is A11122, Invitrogen.
I used protein G-dynabeads.
I followed conventional protocol of IP excepting using cell lysates but mixing 2proteins.
During IP, all buffer is PBS w/ 1% Empigen (detergent))

What are you using for your ECL detection? If you're using a very sensitive detection method you will likely still see the heavy chains even though the antibodies shouldn't be cross reacting. This happened to me, and even when I tried crosslinking the antibody to the beads, the heavy and light chains were still detectable with the ECL kit I was using (i didn't have any choice because I needed the sensitive kit for my antibody). You can try a sample run, just add the antibody to a mix where you don't have anything to pull down and see if it's the heavy and light chains that you're seeing.

[HLee]

I used Super Signal from Pierce. It's more sensitive than ECL from GE.
But, 100% input myc is much weaker than those unknown two bands..
Did you also use Super Signal and solve the problem w/ ECL from GE?

And I will do the control experiment that you mentioned, first.
Thank you!!

smu, on Jun 15 2009, 04:17 PM, said:

HLee, on Jun 15 2009, 12:21 PM, said:

Hi,

I'm doing IP with 2 purified protein to see their interaction.
One is tagged w/ GFP and the other is w/ myc.

I IP w/ aGFP ab (rabbit) then blotted w/ a-myc ab (mouse) or a-GFP ab (same one used at IP).

Blotting /w a-myc ab,
I can see very strong 2 bands at ~ 25 kDa and 53kDa.
25 kDa band is more than 5 times stronger than 52kDa
100% input of myc-tagged protein (~33 kDa) shows much weaker (<1/10) slgnal than those 2 bands.
100% input of GFP-tagged protein didn't show any signal.

Blotting w/ a-GFP ab,
IP shows strong 3 bands at ~ 25 kDa and 53kDa (here, IgG light and heavy chain, absolutely) and 28 kDa GFP-tagged protein
52 kDa bans is ~ 3 times stronger than 25 kDa

With their size, two bands blotted w/ a-myc seems like IgG. But it doesn't make sense at all.
How a-mouse secondary antibody detects antibody from rabbit.
And the patterns of signal strentgh of two bans are differen from blots w/ a-Myc and a-GFP ab.

But, I just mixed 2 PURIFIED protein for IP.
So, in the tube there's no proteins other than 2 proteins and a-GFP antibodies.

Anyone can think what's hapenning, what are those two bands, and ** how to remove those bands!!!!

(a-GFP ab is A11122, Invitrogen.
I used protein G-dynabeads.
I followed conventional protocol of IP excepting using cell lysates but mixing 2proteins.
During IP, all buffer is PBS w/ 1% Empigen (detergent))

What are you using for your ECL detection? If you're using a very sensitive detection method you will likely still see the heavy chains even though the antibodies shouldn't be cross reacting. This happened to me, and even when I tried crosslinking the antibody to the beads, the heavy and light chains were still detectable with the ECL kit I was using (i didn't have any choice because I needed the sensitive kit for my antibody). You can try a sample run, just add the antibody to a mix where you don't have anything to pull down and see if it's the heavy and light chains that you're seeing.