Dear Friends,
Hope you all have fun with research. I wanna do western blot for BDNF from rat and human plasma and serum. Could anybody,if have experience, tell me the dilution factor for serum/plasma, and PAGE and western blot protocols (howmuch micrograms of protein that we need to use on PAGE,buffers,blocking conditions). I need it badly!!!Thanks in advance.
protein load you will need to optimize depending upon the expression of your antigen and the sensitivity of your detection system. For example with ECL advance from GE amersham 10 ug total protein load also detects the antigen of interest. Blocking conditions can vary from 2-5% NFDM or blocking reagent supplied with ECL advance kit. washes can be done with PBS containing 0.05% tween 20. good luck
thanks a lot Ruchi!!! 'll use ur ideas. I'll let u know if I get nice result... :)
Hey Soundara how did this western blot work out for you? I'm trying to run a W.B with rat plasma for GDF-8 and am getting huge Albumin and IGg bands.
Dear Paige, i did wb with rat plasma for low mol wt prtns like bdnf and cytokines which are less than 30kda, and it came good. dilution: 1:2.5 with 1x pbs. i too got strong albumin bands, but l used least dilution for primary antibody to get neat band just to confirm even loading. so my point is that if mol wt of ur candidate protein is less than 50kda, u need not worry. but,if it is about 55 or 60kda, u'll b in trouble dear. and yes, i used 50ug for loading which given strong albumin and igG. so u can try loading less amount like 20 or 25 ug {this helps when ur protein generally presents in large amount in plasma}. in this way, u may have a chance to get ur protein with less albumin background. hope it will help u. gud luck. lemme knw d result also :)
Hello Soundara, I would love to hear how your BDNF WB details. I've done WB for BDNF on the rat but I used an Ab for proBDNF and I worked with whole brain lysates. My group made a BDNF KO rat with ZFNs. cheers
Dear Aaron, I did WB for pro/mature BDNF from serum/whole brain lysate/prefrontal cortex alone with santacruz Abs. Pro/mature BDNF worked in serum but pro BDNF did not work in tissues, all the time!! no problem with mature BDNF I came to know from my Prof that the conversion rate of pro to mature BDNF is very rapid and would be difficult to get pro BDNF in WB (in serum you would find it). I wonder why you have used pro BDNF Ab in BDNF KO rat model (may be you knocked out mature BDNF DNA fragment sequence alone or wanted to confirm the KO or what?)
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