I need help
I am doing a western with secreted protein and I got a strong protein aggregation around 75-80 KD
I also run a lysate sample which does not give the same problem
What can I do to avoid aggregation?

thanks

When you say aggregation do you mean there is a blur on the western at 75-80 kD? Is the secreted protein purified in any way? Protein-protein interactions are generally broken by boiling in SDS-cracking buffer with DTT or BME for 2min. If its aggregation this will take care of it. A blur on the western blot on the otherhand could be caused by 1) degradation 2) glycosylation for starters. Thats a start, fill us in on the details and maybe we can ge to the bottom of things.

Rb

Thank you very much for your help
I will give you more details: I would like to detect a protein that is supposed to be secreted under certain stimulation.
After 24h of treatment, supernatant was collected and total protein extracted from the pellet. Secreted protein sample was not purified or manipulated in any way. Apportion of supernatant was concentrated using a microcon Millipore devices (for 30KD protein).
Samples (Surnatant, surnatant concentrated and lysate) were boiled for 4 in lamely buffer (which contain SDS and BME) and loaded in a gel. After the electrophoresis both REd ponceau staining and WB showed a big intense band around 75-80 KD in secreted protein lanes (supernatant and supernatant concentrated). Lysate lane showed the regular protein pattern. Since this big band was found also in the concentrated supernatant, which is supposed to have protein of 30KD or lower size, I thought about an aggregation.
Probably a supernatant manipulation is necessary to avoid this problem.
I hope my letter is clear.

Thank you in advance

Barbara

Sorry for the slow response,

Im a little confused. Is your protein less than 30kD?

I suppose you took the supernatant from the cells, then concentrated it. did you keep the top or the flow through? the big stuff stays in the top, so you must have taken the bottom fraction and ran that on the gel for the concentrated sample.

My guess is your protein is glycosylated if thats possible let me know, I can give you some advice as to how to proceed.

Rb

I still have problems with secreted proteins!
I prepared new sapmles and still got only one band at 75KD
I tried adding lysis buffer containing detergents or adding triton X-100 but nothing is changed.
As you mentioned the problem could be glycosilation and not aggregation, if this is the case how I can solve it?

thank you in advance

bb

If your problem is glycosylation. It can be genrally be removed by treating with PNGaseF, which specifically cleaves N-linked sugars. Some expression systems add unwanted sugars, yeast is partucularly notorius for this as well some E. Coli strains.

Im still unclear as to the expected size of your protein? And whether this is naturally secreted protein or one you are trying to express off a plasmid.

Also consider that many proteins do not migrate on SDS-PAGE gels at their predicted size. For instance I work on two proteins that have MW of less than 50 kD, but they run as high as 80 kD on SDS gels. So really make sure that the 75 kD band is not what you are really looking for in disguise.

Best of luck
Rb

I forgot to add others on thius site have had good success with the EndoH kit from NEB to remove glycosylation.

Rb

Hi!
I have the same problem: while performing western on my supernatants i'm getting one big band, like there was no seperation of proteins.
What can be the solution for this problem??

Thanks a lot for any answer

Mevka

Same question for you as before. If you Coomassie stain a gel what do you get lots of bands or just one blob?

Rb

Hi

I tried the deglicosilation and unortunately I did not solved my problem. I still have one very big band around 75KD and very few weak bands as before.

Sorry I see above you stained with Ponceau and saw the same band.

You never told me what transfection system you are using and how do you know your "protein" is 30kD.

Rb

I will attach red ponceau picture of the gel rtansfered on nitro so you can see how it look. I loaded different condition of deglicosilation try.

I am not performing any transfection.
I am looking for a natural secreted protein which is going to be secreted following stress stimulation.
My cells are suppose to be a natural positive control for this protein.
I performed different experiment and I was able to see an increase of expression by semiquantitative PCR and by WB in the lysate cells (althogh the signal is dirty due to goat antibody).
Unfortunately I don't detect anything in the supernatant . I need have this WB working for my project, it is crucial and I have no clue anymore!

thank you for your help

Ok now we're getting somewhere.

What are your media conditions? serum/no serum? The reason I am asking is albumin is such a major contaminant in serum that it distorts gels and can cross react with a lot of antibodies. How are you loading the "supernatant" which i assume is the cell media?

You have another antibody that detects the correct band in cell lysates?
Does that antibody detect the 70kD aggregate as well?

Maybe you can try using the goat anibody to immunoprecipitate your protein then detect it with new one.

Rb

I used different media conditions (from 1% to 10% of BSA or human albumin) and I always got the fat band. The only difference was a lower number of small bands detected in 1% serum media samples compared to 10% serume samples.

After collection of media/upernatant I spin down at 10,000 xg for ten minutes. I load into the gel after mixing with 2X leamly loading buffer (containing BME and SDS) and boiled them for at least 5 minutes.
I tried increase time of boiling and denaturing agent concentration but I did no get any difference in the band pattern.

Unfortunately exist just one antibody avalaible and it is in goat, which is giving me preatty dirtiy WB. this antibody is working in detecting the protein in the cell lysate sampe but doesen't detect anything in the supernat samples. (experiemnt don with all samples in the same gel)

thanks