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Fluorescent Western Blotting Help


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#1 _sgrub

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Posted 12 April 2011 - 07:19 AM

Recently we've been trying to optimize a method for immunodetection using secondary antibodies conjugated to fluorophores based on various protocols from the BioRad website (we recently purchased a Bio Rad Versa Doc 4000 MP). Unfortunately, we have had very little success in doing so, aside from detection of protein ladders and a very faint GAPDH that I have uploaded. I was hoping someone might be able to assist us in troubleshooting this problem.

In order to optimize our procedure, we purchased new secondary antibodies conjugated to DyLight fluorophores (of various species specificity and excitation wavelengths), a fluorescent western blocking reagent (blok-FL buffer from Millipore) and Immobilon-FL PVDF (also from Millipore), a low auto-fluorescence membrane. However, after primary and secondary incubation, all the required washings and drying the membrane, we were unable to detect any phosphorylated, total or phosphorylated and total p38, NFkB or JNK on three respective blots for each protein. Staining of the PVDF membrane with Ponceau S solution indicates that protein from the lysates were transferred properly.

Bio Rad tech support has suggested that the antibody concentrations require optimization. Just last week, I used the same primary antibody concentrations for chemilluminescence without error. After a couple of weeks of back and forth with both Bio Rad and the manufacturer of the secondary antibodies, we have confirmed that our imager is working properly and that we are using it properly.

The GAPDH antibody that we use is VERY strong with chemiluminescence, which is why I'm not exactly considering this a success. Our NFkB, p38 and JNK antibodies (both phospho and total) are also relatively easy to image, yet we could not detect any using secondary fluorescence. I guess what I'm asking is, is it wrong to assume that the same primary antibody concentrations should be utilized for chemiluminescence and detection with a secondary fluorescent antibody? Is this problem as simple as adding a large concentration of primary antibody? I have tried a large range of secondary concentrations from 1:3000 to 1:20,000 and have had similar looking blots show up.

Another question I had was concerning the concentration of Tween-20 in our wash buffer. We use TBS with 0.01% Tween, but I have read that Tween can interfere the antigen/antibody interaction.

GAPDH_488_10 sec.jpg

I realize this was a long winded post, so thank you to anyone who took the time to read this and/or might have any suggestions.

#2 _sgrub

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Posted 12 April 2011 - 09:20 AM

It occurred to me that I should have been more specific about my methodology.

1. Samples loaded with protein ladder on Criterion 4-15% Gradient gel at 150 V for approximately 1.5 h
2. Gel removed and equilibrated with Transfer Buffer containing 20% methanol for 15 min
3. PVDF submerged in 100% methanol for 30 sec; equilibrated in Transfer Buffer for 5 min
4. Transfered at 4 degrees at 100 V for 1 h
5. Blot blocked with blok noise cancelling reagent for 1 h at room temperature
6. Blot incubated with primary antibodies at 4 degrees overnight
7. 3x5min wash with TBS-T
8. Blot incubated with secondary antibodies for 1 h at room temperature (After this point, blot handled in the dark)
9. 3x5min wash with TBS-T
10. Blot submerged in 100% methanol for 2 min
11. Blot allowed to air dry for approximately 1 h
12. Blot imaged

#3 mdfenko

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Posted 12 April 2011 - 09:49 AM

it sounds to me that your secondary antibody is the problem. the concentration of primary seems to be okay.

you can test the secondary antibody by dot blotting using primary antibody as the antigen.
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#4 _sgrub

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Posted 12 April 2011 - 11:15 AM

it sounds to me that your secondary antibody is the problem. the concentration of primary seems to be okay.

you can test the secondary antibody by dot blotting using primary antibody as the antigen.


After my initial post, I spoke with an employee at the local university who is having similar trouble with the same process/piece of equipment. He said that after a gel-->PVDF transfer, he soaked the gel in ddH20, methanol then allowed the blot to air dry, and dotting exactly as you suggested. He then blocked the blot as one normally would, did all of the appropriate incubation/wash steps and then imaged to find that the dot from the primary was visible. One can conclude from this that the secondary is binding to the primary and is illuminating properly.

Thank you for the suggestion though, I am going to try this tomorrow morning.

#5 _sgrub

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Posted 13 April 2011 - 12:13 PM


it sounds to me that your secondary antibody is the problem. the concentration of primary seems to be okay.

you can test the secondary antibody by dot blotting using primary antibody as the antigen.


After my initial post, I spoke with an employee at the local university who is having similar trouble with the same process/piece of equipment. He said that after a gel-->PVDF transfer, he soaked the gel in ddH20, methanol then allowed the blot to air dry, and dotting exactly as you suggested. He then blocked the blot as one normally would, did all of the appropriate incubation/wash steps and then imaged to find that the dot from the primary was visible. One can conclude from this that the secondary is binding to the primary and is illuminating properly.

Thank you for the suggestion though, I am going to try this tomorrow morning.


Blot 6 AlexaFluor 488.jpg

I dotted 5 g/ml, 0.5 g/mL and 0.05 g/mL GAPDH from left to right and incubated with secondary antibody conjugated to AlexaFluor 488 at 1:3000 and this is the image I acquired after a 90 second exposure. This image is still less than desirable, but it proves that there is an interaction between primary and secondary.

However, below where GAPDH was blotted, NFkB was also blotted over a similar range of concentrations, yet nothing showed up. I used a secondary antibody conjugated to AlexaFluor 568 at 1:3000.

I also did the same blots with secondary antibodies conjugated to DyLight 488 (GαM) and DyLight 649 (GαR) and the blots were less intense than with the AlexaFluor.

#6 mdfenko

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Posted 13 April 2011 - 12:25 PM

did you try blotting the primary antibodies, rather than the antigen for the primaries, as antigen for the secondary antibodies? this way you will know if the secondary antibodies are good.
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#7 _sgrub

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Posted 14 April 2011 - 04:30 AM

did you try blotting the primary antibodies, rather than the antigen for the primaries, as antigen for the secondary antibodies? this way you will know if the secondary antibodies are good.


I should have noted that in my last post, I did not actually have any purified antibody to use in blotting, so I only used primary antibodies as my antigen, for both GAPDH (anti-) and NFkBp65 (anti-).

#8 mdfenko

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Posted 15 April 2011 - 11:36 AM

are your primaries made in the same species?

if so, are they the same subtype?

are your secondaries made against a specific subtype?
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#9 _sgrub

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Posted 18 April 2011 - 04:34 AM

are your primaries made in the same species?

if so, are they the same subtype?

are your secondaries made against a specific subtype?


Anti-GAPDH
Mouse IgG-kappa

Goat anti-Mouse DyLight 488
Mouse IgG- whole molecule

"This antibody will react with heavy chains of mouse IgG and with light chains of most mouse immunoglobulins"


Anti-NFkB
Rabbit- the datasheet does not specify which antibody subtype

Goat anti-Rabbit DyLight 649
Rabbit IgG- whole molecule

"This antibody will react with heavy chains of rabbit IgG and with light chains of most rabbit immunoglobulins"

The secondary antibodies are not targeted toward a specific subtype of antibody. I appreciate all of your help mdfenko, but I think I'm going to put the validation of this method on the back burner for a bit. I have other work to do and my boss thinks I shouldn't necessarily focus on this method if we can still image with chemiluminescence. I am going to contact the company again and see what I can come up with/will post some results for others if I figure out the problem. Thanks again.




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