I am working on AD using drosophila model system.
I want to go for the western blotting from the head region of drosophila, So can anyone help me by providing the following details-
1. what should be the amount of protein I should load.
2.what should be the gel running condition (Volt and ampere)and how much %age of gel.
3.what should be the transfer condition (volt and ampere)
4.I have only .45micron pore size PVDF membrane. Is it suitable for my experiment or less pore size should be used. I want to used separate 4-16 KDa protein.
It will probably take you a few attempts before you find the optimum conditions for WB and these conditions will vary depending on what protein you are looking at, it's size and even expression level. Below are some comments that may help...
1. Amount of protein - I usually use 6 heads for Drosophila WB. More than this over saturates my particular gels and I get smears. The amount you need to load will depend on your gel thickness and well size. So it is worth doing a test gel where you load protein extract from 1 head, 2 heads, 3 heads etc in the wells, to see which is best. You can even do this on WT or w1118 flies and probe for a ubiquitous protein like B-tubulin if your experimental flies and antibody are too precious to use up. If you need to know the protein concentration for subsequent analysis (for example), you can then carry out a protein assay on the optimum number of heads. (ie RC DC).
2. The percentage of the gel depends on the size of the protein you wish to detect... so, what size is B-amyloid? For smaller proteins, you generally need a higher percentage gel. Ideally, you want the protein of interest to run to the lower half of the gel. This document has lots of useful info http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040A.pdf . The amount of time you run the gel for will depend a little on the gel percentage. You want to run the gel so that the loading dye just reaches the bottom of the gel. I would pick between 150-200V and keep an eye on the gel, but it may take between 30mins-1 hr. Again, you may want to do a few test gels run at different voltages.
3. Transfer - smaller proteins transfer faster than larger proteins. Try 1hr at 100V. Reduce the transfer time if it looks like you have lost some protein.
4. I do not often use PVDF membrane so cannot offer much advice on this, but the smaller pore size will 'trap' more protein. I think 0.45 should be ok. If you think you are losing protein because it is passing through the membrane, reduce the transfer voltage.
Hope this helps.
The answer depends on the protein you want to look at, not what region it is extracted from. I use for whole body or parts of it 38 min, 200V for running the gel, 1:20 hours, 100V for transfer. That works fine for the proteins I am interested in.
I am looking for the beta amyloid protein from Head region of drosophila.
It will probably take you a few attempts before you find the optimum conditions for WB and these conditions will vary depending on what protein you are looking at, it's size and even expression level. Below are some comments that may help...
1. Amount of protein - I usually use 6 heads for Drosophila WB. More than this over saturates my particular gels and I get smears. The amount you need to load will depend on your gel thickness and well size. So it is worth doing a test gel where you load protein extract from 1 head, 2 heads, 3 heads etc in the wells, to see which is best. You can even do this on WT or w1118 flies and probe for a ubiquitous protein like B-tubulin if your experimental flies and antibody are too precious to use up. If you need to know the protein concentration for subsequent analysis (for example), you can then carry out a protein assay on the optimum number of heads. (ie RC DC).
2. The percentage of the gel depends on the size of the protein you wish to detect... so, what size is B-amyloid? For smaller proteins, you generally need a higher percentage gel. Ideally, you want the protein of interest to run to the lower half of the gel. This document has lots of useful info http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040A.pdf . The amount of time you run the gel for will depend a little on the gel percentage. You want to run the gel so that the loading dye just reaches the bottom of the gel. I would pick between 150-200V and keep an eye on the gel, but it may take between 30mins-1 hr. Again, you may want to do a few test gels run at different voltages.
3. Transfer - smaller proteins transfer faster than larger proteins. Try 1hr at 100V. Reduce the transfer time if it looks like you have lost some protein.
4. I do not often use PVDF membrane so cannot offer much advice on this, but the smaller pore size will 'trap' more protein. I think 0.45 should be ok. If you think you are losing protein because it is passing through the membrane, reduce the transfer voltage.
Hope this helps.
I am still waiting some more suggestions, please leave your valuable comments.
my interest of protein is 4kDa. and I am using nitrocellulose membrane of .2 micron
You can load 15-20 micro-litre of protein extract per well. Since your size of protein is smaller you can use 12%-14% resolving gel. I normally run the gel at 100-120 V and transfer at 320mA for 2hrs in cold room.
Hi I am using heads for my Westerns with Drosophila and I am looking at the MAPK proteins. I use 20-25 heads for preparing my protein extracts with SDS sample buffer. I load 20ug of protein for the small gel, run it at 50V set the current to maximum for half an hour, then at 100V for 1.5-2h. I transfer at 100V for 1.5h. It works fine for me. My protein is 37 kDa so this protocol works for me.
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