4 replies to this topic

[noisette1325]

hello,

i want to make a western blot of gamma-interferon in order to estimatate glycosylation pattern of it. That is non glycosylated form has a MW of 17 kDa, monoglycosylated 20 kDa and biglycosylated 25 kDa. I have found old laboratory notebook with the western blot I would like to make. But my signal is very weak. After optimisation of transfer condition, I obtained something like this.


The bands have a molecular wheight slightly more than 25 kDa. I hesitate if this is the biglycosylated form of if it is a dimer. In the former western, I was able to distinct the three form with the dimer form. However, the insensity was to low to interpret this.

So the problems are:
-the unrepetability
-the weak signal
-the denaturation (if the sample is denaturated, why are dimers observed?)
-the continuous band between the sample. (on the photo, you can see that the intensity is higher between the lane than in the lane).

thank you very much for all advices.

[noisette1325]

I forgot my experimental conditions.
warm denaturation 10 minutes
transfer with 25 V , 20 minutes. I try also 10, 30, 40, 80 minutes. 20 and 40 minutes is done with 1 and 2 membrane. Transfert buffer is methanol 20% and SDS 0,5%
Blocage buffer : TBS-tween + 5% milk
Fisrt antibody is diluted 1000x with tween + 0,5% milk (producers propose 50 000x)
Second antibody is diluted 3000x.
Dillution and washing with TBS (I'll try PBS).

[mikew]

#1. Do a negative control. If you are using antibodies for the first time and even the 100th time, use a cell line that should give you a negative result. This shows antibody specificity.
#2. Aside from some old lab book, why do you think you get multiple bands for IFN-gamma?
Are there examples of this in the literature?
For example, this paper shows a IFN-gamma Western and it appears as a single band.
British Journal of Cancer (2007) 97, 420–425. Li, Zhang et. al  
Expression of interferon- gamma in human adrenal gland and kidney tumours

[noisette1325]

I have partly resolved my problem. There was an insufficient transfer of the protein.

I'm working with cells cultivated in suspension that have been transfected with the IFN gene. Cells in culture have not the same glycosylation pattern than living tissue. You can look at this paper

Fortification of a protein-free cell culture medium with plant peptones improves cultivation and productivity of an interferon-gamma-producing CHO cell line - Burteau et al, 2003, In vitro.

There is several bands.

[mikew]

Well, good luck.
I looked at that paper and they indeed get multiple bands.
I am not sure if these are due to the antibody being of poor quality.
Alos, the antibody they use from Pierce has not been published to recognize IFN-gamma
by Western. I am dubious about the glycosylation of this protein leading to multiple bands and it not merely being an artifact of a crappy antibody.
  It's interesting that Pierce has about 10 references for ELISA assays and not even 1 for a Western for this antibody.
Hopefully I'm wrong....