Western Blot - Loading Control Problem

[BevB]

Hello,

this is an unusual problem, but there may be someone out there who has been in a similar situation.

 

I have an experiment where I induce hypoxia on cell samples, collecting them at specific time points over 24 hours, and measuring a particular connexin level in them.

I measure the protein concentration of the samples using a Bradford Assay, to ensure that I have the same level of protein in all my lanes.

 

In my control samples (no hypoxia), my housekeeping antibody (alpha tubulin) levels are pretty similar over the 24 hour period, reassuring me that I do have similar levels of proteins in my lanes.

 

However, in my hypoxic samples, this is not the case. The levels of alpha tubulin rise progressively over the 24 hour time period, looking like I have not considered protein concentration over time.

 

Is there a slim possibilty that my house keeping antibody is being affected by the hypoxic nature of my experiment? I have heard that alpha tubulin is a pretty reliable house keeping antibody, so I don't know if this will be the case.

I have triple checked my calculations of my protein concentration (and will be doing another Bradford assay in the near future), but I really don't think that I have made a mistake here.

 

If anyone else has come across their housekeeping antibody being affected by their experiment, please could you throw some advice this way!

I guess one obvious option that I have is to change my housekeeping protein - but I don't want to run into the same problem.

 

Thanks in advance

Bev

[CharonY]

First, I am not familiar with the effects of hypoxia, so consider my response in this context. However, house-keeping genes are rarely stable under each condition (especially during stress). This is something that has driven qPCR guys nuts. My question is therefore whether there is data to indicate that alpha tubulin is a suitable reference protein during hypoxia.

 

You may want to check this out:

J Matern Fetal Neonatal Med. 2004 Jul;16(1):55-9.

The effects of hypoxia on growth cones in the ovine fetal brain.

Morgan BL, Chao CR.

Source

For normalization purposes I therefore usually first test whether the reference (in this case house-keeping protein) is indeed stable throughout the experiment.

[Dr. Monte Miller]

I have actually worked with Hypoxic cells and in fact worked with HIF-1, hypoxia inducible factor 1. I couldn't agree more that you most likely need to change your housekeeping protein. Look up HIF-1 alpha on pubmed and you are sure to find an article or 2 that could give you a good housekeeping protein or a protein that does not change concentration under your conditions.

 

mmiller@forensicdnaexperts.com

[shaung]

maybe compare beta actin levels with other house-keeping genes such as GAPDH.