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Detection of T7 RNA polymerase by WB


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#1 pDNA

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Posted 17 October 2012 - 09:17 AM

I have problems detecting T7 RNA polymerase in e.g. samples from E. coli BL21(DE3) after induction.

I use a primery (mouse monoclonal T7 RNA Polymerase antibody [IgG1]) and secondary (Goat Anti-Mouse IgG [H+L] AP Conjugate) from Novagen for the detection of the T7 RNA polymerase. Unfortunately so far without success!

I tried dection with BCIP and CDP-star already ...but was not able to get any real signal yet. My positive control is stained but i'm not able to detect anything in my samples. My samples have to contain T7 RNA polymerase since i can detect a load of my recombinant protein in my samples.
I do lyse my cells and then fractionate into soluble and insoluble fraction. I normally load both fractions on a gel. In the original protocol they do the use the whole cell extract. Can this make a difference?

Has anyone an idea what could be the reason for my misery?
Are there alternatives available to detect T7 RNA polymerase?

Best regards,
p

#2 Curtis

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Posted 17 October 2012 - 02:30 PM

My samples have to contain T7 RNA polymerase since i can detect a load of my recombinant protein in my samples.


you mean you see a band on your sds-page gel?

#3 pDNA

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Posted 17 October 2012 - 09:38 PM

yes, i'm able to see my recombinant protein (that i express using T7 RNA polymerase) on the SDS-PAGE gel ...but i'm not able to detect the polymerase itselfe ...what makes me kind of crazy!

Regards,
p

#4 Curtis

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Posted 18 October 2012 - 09:04 AM

That's strange since your positive control is stained. It can't be that your antibody isn't binding. It must be the sample itself.

#5 pDNA

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Posted 18 October 2012 - 09:45 AM

The Novagen protocol says:
"Using 1/50 of the total cell protein from 1 ml of an induced λDE3 lysogen will generally give a strong signal."

I always use 3/OD (that gives me approx. 1 mg cell dry mass, at OD 0.6 i take 5 mL, pellet and lyse) ...i resuspend the soluble and the insoluble fraction in 200 µL and load 12 µL on a gel ...that corresponds to ~1/40. I really have no clue why i'm not able to get any signal.

Is it really that hard to detect low level expression genes from whole cell lysates?

Regards,
p

#6 Curtis

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Posted 18 October 2012 - 05:51 PM

You know, I just remembered that sometimes I used to see my IPed samples on the gel too, but I couldn't detect them on the membrane. Then I changed my Towbin's buffer and played around with the time of electroblotting. I also activated my PVDF membrane for 1 min before soaking it in Towbin's buffer, and then it appeared on the membrane....I am sure you already know how to handle this, sine I know you for a long time...I also found that 30-60 ug of total protein is enough to load on BioRad's Protean Mini 3. less than 10 ug was really not detectable.

The first thing that came to my mind about your case was that maybe your antibody doesn't label your protein. But then you mentioned that your positive control is stained. Now, is it possible that your control and your sample have different morphology? sometimes when few amino acids are different the antibody cannot bind to the protein. Maybe you need to try a polyclonal antibody?

#7 pDNA

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Posted 18 October 2012 - 10:50 PM

Dear Curtis,

many thanks for your input so far!

I thought about that blotting issue as well ...but my standard is blotted very well (even the 200 kDa band), therefore i believe that "all" the proteins get transfered (you never know).

Maybe my problem is due to the fact that i seperate soluble and insoluble fraction and in the original protocol they use whole cell lysate. Maybe the T7 RNA polymerase is attached to some cellular structure that gets lost during fractionation? I have no clue.

I will try whole cell lysate and see if i'm able to detect the protein.

Regards,
p




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