Author Topic: diffrence between 0.2 micron and 0.4 micron membranes  (Read 7576 times)

NA

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diffrence between 0.2 micron and 0.4 micron membranes
« on: April 11, 2012, 12:02:40 AM »
Hi,

I just wanted to know that when we should use 0.2 micron membrane and when 0.4 micron.Currently I am using Biorad's  Immuno Blot PVDF Membrane(cat #162-0177) for both high mol wt protein(PARP) and low  mol wt protein(BCL2) from rat kidney tissue getting good bands for BCL2 but struggling with PARP.Protein transfer is very good, tried antibodies from different companies including cell signaling,extraction buffers ,using Pierce west pice substrate but nothing working.either there is no signal or two much non specific.Kindly suggest me what should I do and is the mebrane is causing any problem itself.

Susanne GE

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Re: diffrence between 0.2 micron and 0.4 micron membranes
« Reply #1 on: April 16, 2012, 03:18:40 AM »
Hi, 0.2 micron membranes are mainly recommended for small proteins while 0.45 micron membranes are recommended for medium to large proteins. In your case it is unlikely that your problem to detect PARP is related to choice of membrane since the protein size is 116 kDa. In general PVDF membrane has higher protein binding capacity than NC membrane and is recommended for best detection sensitivity. I would rather think that your problems to detect PARP is related to that you have to low amount of the protein or that the Ab is not good enough. I suggest following
1. If possible load larger protein amount to increase the PARP amount. it is important to have the protein in detectable levels.
2. Secure that the Ab you use is aimed for Western blotting and if supplier show any WB data of the Ab performance. it is also impAb ortant to optimize the dilution factors.
3. Use a more sensitive detection reagent, the West Pico reagent has typically a medium detection sensitivity and there are other reagents with higher detection sensitivity available. I would strongly recommend you to try Amersham ECL Select, a new very high sensitive reagent from GE Healthcare. If you are interested to try please contact a sales rep for GE healthcare, there are trial sample kits for free available.
4. To avoid/decrease unspecific bands it might be worth to evaluate some other blocking agents. Blocking agents works very different for different proteins and there is no one blocking that is optimal in all blottings.
5. Secure that the transfer efficiency is good in the higher Mw area, you can stain the membrane with Ponseau-S after transfer to check that you have an even transfer or use an pre-stained colored marker to check the transfer efficiency.
It might be necessary to increase the transfer time if the larger proteins do not transfer effficient. If this cause problem with the small protein then, it might be possible to cut the gel in two pieces and transfer them separately, one optimized for the small protein and the other for the large protein.

I hope these suggestions helps.

Kind regards
Susanne
I hope this suggestions and recommendations helps.