Hi Tasfia,

Here is a link to the protocol page from abcam. It is quite comprehensive and if you follow the guide you shouldn't have too many issuse. But once you begin, if you have any specific questions I am sure people will be more than willing to offer some advice.

There should be antibody specific information with each antibody that you buy, so also read the product sheet for you cytokines, and that will let you know if anything special is needed for for detection.

http://www.abcam.com/index.html?pageconfig=resource&rid=11375

Hi Tasfia. I usually detect cytokines using multiplex beads, which is quite accurate, but assume you may not have access to this technique. From my experience in western blots, the most sensitive detection is using biotinylated antibodies and streptavidin. But be careful with the background. If your cytokine target is abundant, it may be preferable to use a less sensitive reagent, so as to avoid excesive background. Blocking is quite important, do not use milk powder for this if you decide to use biotin in the detection, because milk may contain biotin too.

Overall, you avoid ´smiling´ in the separation step by using a moderate voltage and fresh buffers.

Small guide I like to give to students in the lab attached to the post.

All right and credit to Millipore/Merk

RD also have a nice guide

http://www.rndsystems.com/western_blot_detail_objectname_qc_protocol.aspx

Hallo, I would suggest to quantify cytokine by EIA assay (o ELISA). EIA assay by cayman are very simple!

Hello everyone.

Thank you very much for your very helpful suggestions. I will look into the suggested guides.

Carla, yes, I did perform ELISA on them; I was basically looking for alternative methods. Moreover, I work with nanoparticles, and I want to rule out the possibility that nanoparticles were interfering with the assay. As the PAGE would separate the nanoparticles, I wanted to use the western to validate my results.

Hi Tasfia, 

if you think nanoparticles may interfere with the ELISA, just add some nanoparticles to some of the wells and see whether you get any background noise. I think the best way is still the ELISA, its much more quantitative than the wetern blot.  However, if you just want to confirm, why dont you just do qPCR?  its much cheaper than the western and you dont need as much sample.

Tasfia, We use Gel Doc and versa Doc for detection. Versa Doc is good in the sense that it defines the boundaries of the membrane. In case of Gel Doc, It only displays the boundary if there is any blot (at times we did not have anything on the membrane so gel doc was not able to show the whole membrane). For western blotting, the most critical step is transfer. You will have to optimize time according to the weight of your protiens. We, for a 66 KDa protein, give 105 minutes, using semi dry blot from thermo scientific. For 180 KDa protein we usually give 2 hours. If you are not able to detect bands, start a check from secondary antibody first. Secondary antibody dilution gives best results with in two to three times use. So, you will have to make fresh solution as you start getting dim or light bands with the same secondary antibody dilution. Another point to consider is the amount of protein. We use 3X10^5 cells to get the desired protein. If your protein is abundant, you can use less amount of cells. If it is meagre, you will have to use more cells. I hope that will work for you. All the best.

Thanks a ton, Ruchi.