4 replies to this topic

[fortunate]

A veteran Western Blotter encountered a new problem
Please take a minute to have a look at my uploaded image.

My purpose is to check is a GST fusion protein interacts with my target protein Erk1/2. So I used GST or GST fusion protein bound to glutathione agarose beads to pull down Erk1/2 from cell lysate. Then probe with anti-p-Erk1/2 antibody.

The puzzle is what caused the bubble like bands? I am sure they aren't air bubbles because a separate membrane with a different antibody showed same band pattern.
They are not from over-exposure,because I tried 30 secends exposure and signals are faint.

I suspect bubble bands are my GST fusion protein.Is so, how can I get around this? It mocks my intended Erk bands.

Praying for replies!

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[casandra]

fortunate, on 27 July 2011 - 02:45 PM, said:

A veteran Western Blotter encountered a new problem
Please take a minute to have a look at my uploaded image.

My purpose is to check is a GST fusion protein interacts with my target protein Erk1/2. So I used GST or GST fusion protein bound to glutathione agarose beads to pull down Erk1/2 from cell lysate. Then probe with anti-p-Erk1/2 antibody.

The puzzle is what caused the bubble like bands? I am sure they aren't air bubbles because a separate membrane with a different antibody showed same band pattern.
They are not from over-exposure,because I tried 30 secends exposure and signals are faint.

I suspect bubble bands are my GST fusion protein.Is so, how can I get around this? It mocks my intended Erk bands.

Praying for replies!

ghost bands? you probably have too much proteins or high concentration of primary/secondary abs that the ECL substrate gets depleted really quickly so no light is emitted after the reaction....try loading less protein or decrease the antibody concentration and see if the white bands will disappear...

[Wost]

Biochim Biophys Acta. 1994 Jun 12;1206 (2):272-8  
Inactivation of horseradish peroxidase by phenol and hydrogen peroxide: a kinetic investigation.
K J Baynton, J K Bewtra, N Biswas, K E Taylor
Inactivation of horseradish peroxidase (HRP) was examined in the presence of hydrogen peroxide alone and in the presence of hydrogen peroxide plus phenol. HRP is inactivated upon exposure to hydrogen peroxide (H2O2) by the combination of two possible pathways, dependent upon hydrogen peroxide concentration. At low H2O2 concentrations (below 1.0 mM in the absence of phenol), inactivation is predominantly reversible, resulting from the formation and accumulation of catalytically inert intermediate compound III. As H2O2 concentrations increase, an irreversible mechanism-based inactivation process becomes predominant. The overall inactivation comprised of both processes exhibits a second-order inactivation rate constant (kapp) of 0.023 +/- 0.005 M-1 s-1 at pH 7.4 and 25 degrees C. In the presence of both hydrogen peroxide fixed at 0.5 mM and phenol, HRP was inactivated in an irreversible, time- and phenol concentration-dependent process, also mechanism-based, with a kapp of 0.019 +/- 0.004 M-1 s-1.

[fortunate]

casandra, on 27 July 2011 - 07:23 PM, said:

fortunate, on 27 July 2011 - 02:45 PM, said:

A veteran Western Blotter encountered a new problem
Please take a minute to have a look at my uploaded image.

My purpose is to check is a GST fusion protein interacts with my target protein Erk1/2. So I used GST or GST fusion protein bound to glutathione agarose beads to pull down Erk1/2 from cell lysate. Then probe with anti-p-Erk1/2 antibody.

The puzzle is what caused the bubble like bands? I am sure they aren't air bubbles because a separate membrane with a different antibody showed same band pattern.
They are not from over-exposure,because I tried 30 secends exposure and signals are faint.

I suspect bubble bands are my GST fusion protein.Is so, how can I get around this? It mocks my intended Erk bands.

Praying for replies!

ghost bands? you probably have too much proteins or high concentration of primary/secondary abs that the ECL substrate gets depleted really quickly so no light is emitted after the reaction....try loading less protein or decrease the antibody concentration and see if the white bands will disappear...

Thank you so much! I plan to titrate my GST-fusion protein. I must have added too much of it in hopes that it can bind so much target protein as I can detect.

[casandra]

fortunate, on 28 July 2011 - 11:56 AM, said:

casandra, on 27 July 2011 - 07:23 PM, said:

fortunate, on 27 July 2011 - 02:45 PM, said:

A veteran Western Blotter encountered a new problem
Please take a minute to have a look at my uploaded image.

My purpose is to check is a GST fusion protein interacts with my target protein Erk1/2. So I used GST or GST fusion protein bound to glutathione agarose beads to pull down Erk1/2 from cell lysate. Then probe with anti-p-Erk1/2 antibody.

The puzzle is what caused the bubble like bands? I am sure they aren't air bubbles because a separate membrane with a different antibody showed same band pattern.
They are not from over-exposure,because I tried 30 secends exposure and signals are faint.

I suspect bubble bands are my GST fusion protein.Is so, how can I get around this? It mocks my intended Erk bands.

Praying for replies!

ghost bands? you probably have too much proteins or high concentration of primary/secondary abs that the ECL substrate gets depleted really quickly so no light is emitted after the reaction....try loading less protein or decrease the antibody concentration and see if the white bands will disappear...

Thank you so much! I plan to titrate my GST-fusion protein. I must have added too much of it in hopes that it can bind so much target protein as I can detect.

yup, a very good idea...you practically have an almost total labelling in those two lanes..let us know how it turns out...