Author Topic: protein diffusion  (Read 2922 times)

acroberts

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protein diffusion
« on: November 04, 2013, 08:58:59 AM »
Hey there. I'm having problems with my low molecular weight proteins. As you can see in the image I attached, the actin and GAPDH bands (though not my target protein) seem to diffuse, making it hard to differentiate between the 9 lanes and making it impossible to quantify. This has been happening repeatedly in many membranes, clearly indicating that there is a crucial step I'm making a mistake in. I stained the membrane with Red Punceau, and lanes are clearly saparated, though they do become wider the smaller the protein is. I'm also attaching the full image for my target protein (not only my band of interest), where you can see non specific signals over the membrane, getting a sense of the lane difusion that is taking place. Do you have any idea of what could be happening?
Thanks in advance.

Susanne GE

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Re: protein diffusion
« Reply #1 on: November 19, 2013, 03:10:51 AM »
Hi, both actin and GAPDH are very highly expressed proteins. To be able to detect a target protein, which usually are present at much  lower levels, you have to load quite much of the lysate. The levels of actin and GAPDH are then very high and the signals from those bands are very strong compare to the target protein signal. It might even be that the strong signals are saturated resulting in in a non-linear respons between protein amount and signal intensity. In your case, I think there is a diffusion, or bleeding, of signal rather than protein diffusion (due to to intense signal). You have to decrease the signal output from actin and GAPDH in order to get reliable quantitation. This may be done by increasing the antibody dilutions. My suggestion is to increase primary and secondary Ab at least 3 fold or even more depending on what dilution you are using right now. My experience is that actin and GAPDH antibodies are very good and you can dilute them up to 1:10000. Recommeneded dilution for the secondary Ab varies depending on what kind of ECL reagent you use. If you use a high sensitive reagent like ECL Prime or ECL Select I would suggest dilutions of at least 1:50000-100000.
Using housekeeping proteins for normalization in quantitative WB has been discussed in different articles lately and it has been show that there is a problem with linear respons when having high total protein amounts (which you usually need to detect the target protein). It is worth to spend some time to check the signal respons and linearity and optimize ab-dilutions in the protein amount range you are working in. I know that GAPDH has a very poor lineraity while both actin and tubulin shows better linearity. Another possibility, which gives a more reliable quantitation, is to instead do the normalization to the total protein amount in the lane. This can be done by labeling of the sample whith Cy3 or Cy5 prior SDS-PAGE and WB. The target protein can then be detected using primary Ab and a Cy5 or Cy3 labeled secondary Ab (ECL Plex). Target protein and total protein can be simultaneously detected using a detection system for Cy5 and Cy3. However, I would start to optimize the ab-dilutions , increase the dilutions in order to get a weaker signal from actin and GAPDH. You may also try tubulin. About the unspecific bands you can try other blockings and/or another primary Ab. Additional washing steps may help and altering high (up to 0,5 M NaCl) and low salt washing steps. But be careful with the high salt to not wash away too much.

I hope this can be to some help.

/Susanne