HSP 104 - (Aug/02/2011 )

Hi you all! I am trying to detect heat shock protein 104 kDa from Candida albicans but I do not get any signal in the membrane. I do notget any signal after the Blot stainning with Ponceau. But anyways I incubated with the antibodies and I do not get any signal. How it is a protein with a molecular weigth >100 kDa I decrease the ethanol concentration to 10% and added SDS 0,1 % (final concentration) to the blot buffer. I still did get any signal ofthe protein. I used a concentration of 0.5 µg mL-1. I do notknow more what to do. Any help???
Thanks a lot

fabipp on Tue Aug 2 20:31:47 2011 said:

you didn't see anything after Ponceau staining? You meant no proteins at all? Is it the first time you're doing PAGE and western blotting? It is better if you give more details of your protocol so the people here can help you troubleshoot...

Hi! Yes, I stained with Ponceau after Blotting and I did not see any signal of the protein. In other Blot I could detect 70 kDa protein, with any problem. So, I was wondering that maybe this high weight proteins are more complex to work with. or should I try just to change the antibody?

fabipp on Thu Aug 4 12:23:20 2011 said:

Ok, before testing or buying new highway robbery antibodies, it would be better to figure out first if that's indeed your problem? Didn’t I ask for more details of your protocol? Ponceau is used for checking the transfer efficiency so it does total staining but it’s not really that sensitive. If your protein is not abundant, you’d probably not see it on the membrane. Are your protein samples crude lysates, extracted or purified? How much did you load per well...perhaps you can still increase the amount. Have you checked for your protein using another assay eg dotblot?

What percentage of gel do you use? For higher molecular weight proteins, it’s better to use lower percentage. You can also try adding 0.05 % SDS in your transfer buffer and lower the methanol to 10%. What’s the pore size of your membrane? Try using 0.45 micron. High MW weight proteins take longer time to transfer so usually, if you stain your gel after a standard transfer, you’d often see some of them still remaining in the gel. You can try increasing transfer time but just take care not to overheat the system.

What dilution of primary antibody did you use? Are you sure it’s working/freshly prepared? You have a positive control that you can test it with? Perhaps you can still increase its concentration or incubate longer.

I did not use dotblot, very good idea! My protein is purified, I also bought to serve as my positive control. I am using 15% acrylamide gel. And I also decrease methanol concentration and add 0.1% SDS (final concentration). The pore size of my membrane I do not know. Iwill check when I am at the lab. How can I stain my gel after the tranference? And how long should I increase the tranfer time, I am usually using 50 minutes. Thank you soooo much for your help.

fabipp on Fri Aug 5 21:30:55 2011 said:

If you already have a positive control, then it's a good way to test your antibody and your technique-how you do your PAGE and immunoblotting. Have you tried just running your sample and simply staining the gel with coomassie brilliant blue? It's not so sensitive- probably it can detect a minimum of 0.5 ug (but you have to check this out) but for most practical purposes it works or you can use silver stain which can be 50 to 100 times more sensitive. At least you'd have an idea if you've really got the protein or how "successful" your purification is.

Also try using 8 to 10% gels instead...and you can definitely increase your transfer time depending on the current you use. We do 100v for 2 hours in the cold and but still some higher MW proteins (250 kDa or more) are left in the gel so you can do your own tweaking and determine which is the best time to stop the transfer.