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fused bands of beta-actin in western blot

beta-actin western blot

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#1 birdyeagle

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Posted 19 June 2013 - 01:23 PM

Hello,
Lately, I am in trouble with getting a beautiful loading control.
I couldn't get clear, separated bands with beta-actin.
However, on the same blot, everything looks OK with other antibodies (ex. CREB, ERK).

All beta-actin bands are connected (fused) together with each other, like hand in hand *__*

Here are the conditions:
Loading 30ug total protein lysate, (boiling before running)
Running at 80v first then 100v,
Transferring at 100v for 1.5hr,
Blocking with the commercial blocking buffer (Rapid block),
1st antibody : 4'C overnight or RT 2hr, (5000x or 10,000x dilution)
2nd antibody: RT 1hr (5000x or 10,000x dilution)

I don't think the problem is on the running or transferring (other target proteins are good),
Blocking buffer? but my colleages don't encounter this problem with Beta-actin (Sigma).
Please kindly give me some advise.

Thank you so~~~ much!

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#2 mdfenko

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Posted 20 June 2013 - 04:51 AM

this problem is occurring during the running of the gel (stain one, you'll see). if you look closely, you will see that the erk also has connections between lanes, albeit not as strongly stained as with actin.

a few factors that can cause this:

sample load- there may be too much protein

time between loading and start of the run- diffusion of the sample will occur

salt in the sample buffer- salts can have strange, unpredictable effects

other additives in the sample buffer (eg detergents, lipids, peg, organics, etc)- (see salt)

gel type- gradients are more prone to this, especially when run slowly you will see more sideways diffusion of the bands, more so with smaller proteins (path of least resistance)

gel concentration (including %c)- restricted mobility

more information on your procedure(s) are necessary to determine why it happens to you and not to your colleagues.
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#3 birdyeagle

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Posted 20 June 2013 - 07:18 AM

Hi mdfenko,

I usually run 30ug of total protein. Beta-actin usually look good at that time.
And we only use regular (no gradient) gels.
Maybe the problem is the salt or additives in the sample buffer as you mentioned, because I used old sample buffer and my colleagues use more fresh sample buffer.

I will try to load less proteins and reduce the time between loading and run today.
Hope it'll get better...

Thank you ~~ ^__^

Edited by birdyeagle, 20 June 2013 - 07:26 AM.






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