Hi,

Abcam suggests Transferrin as loading control for serum. I copy the link:

http://www.abcam.com/primary-antibodies/loading-control-guide

Cheers

D

Thank you very much

I´ll try

Best regards

Christine

Hello,

This article may be helpful for you.

http://onlinelibrary.wiley.com/doi/10.1002/pmic.201200550/abstract

ALL THE BEST!

thank`s a lot for your information

I`ll check it out

Best regards

Christine

Hi Christine.  Loading controls for Westerns of serum are notoriously difficult to choose.  Even the smallest changes in the system can alter amounts of different proteins in the blood.  After an exhaustive search on this issue while I was trying to publish a paper with serum Western blot data from mice (this applies to humans too), I published without a traditional loading control.  Instead I loaded 1uL serum per lane and ran multiple lanes for each sample.  You can see the paper here: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0012909.  The blot is Figure 7D.  This method was accepted by reviewers and is fairly common practice.  Best of luck!

I am not sure what you want to do. I you looking for human antibody in serum or are using the human serum antibodies to stain a specific antigen in cell extract. If you can explain what you want to do maybe I can give you some help.

Best regards,

Janos

Thank you for your answer, Heather!

it`s very helpful and I`ll discuss it in my group.

Thank´s once again

Best regards from Munich

Christine

Dear Janos,

thanks for your interest.

I want to look for human antybodies in serum.

It seems to be quite difficult.

Thank`s once again for your efforts

best regards

Christine

For any WB on serum samples, the difficulty is not the loading control but the high abundance of serum albumin. We standard use a kit for albumin removal (actually, we never get all albumin removed), for sufficient reduction so the albumin-depleted serum will run nicely without any distortions. With such samples, it becomes a lot easier to probe your blot with anything to detect antibodies, or other serum proteins. But please do include a loading control antibody when comparing different samples. The GAPDH antibody should really work once the serum samples have been albumin-depleted.

hej Jan,

thank you for your answer. we already decided to remove the serum albumin with a special kit and THAN we`ll try again. Of course we have 8 different samples.

Thanks once a gain and have a nice time

Christine

Hi Christine,

Even I am trying to validate a human plasma protein using western blot. Initially I thought I will go ahead without the loading controls by just loading equal amount of samples in triplicate (3 lanes). But I am a bit confused seeing few papers with loading control for plasma proteins. Is it mandatory that we have to use the loading control? 

Sorry for asking a question in an answer thread. Thanks for the valuable time and help.

The loading control is not only to satisfy reviewers that all lanes are equally loaded, it also checks for any running and transfer artifacts. It always remains a challenge when you think each lane contains identical protein and still you see differences in intensities of the immuno-labeled bands. When you have your controls in place, and all look identical, at least you can conclude that any variations are not trivial.

My experimental samples create changes in albumin levels as well. I am having the same issues what most of you are facing. Anyone who has more understanding of plasma proteins who can suggest us few good loading controls. GAPDH failed in our lab as well. Tried tubulin and gave a big smear rather than a desired MW band. Tried Actin which did not work either.

I want to first apologize for asking a question to your question.

I know that this is an older thread but I am currently looking for a control to use with human serum samples as a primary antibody. Just curious if you were able to use any of these recommendations with success.