3 replies to this topic

[Zlll]

Hi everyone,

I am a newbie in WB and was trying to analyse the expression of Nav1.8 sodium channel from rat DRG lysates.

I purchased the Anti-Nav1.8 Ab from the Alomone Labs Ltd. (Jerusalem, Israel) as I found quite a number of papers trying to use their product for IHC or WB of sodium channels.

With refer to the data sheet supported with the product, an illustration of Western blot analysis of rat DRG lysates has been shown. The Anti-Nav1.8 Ab is shown to recognize two visible bands at 250kDa and 100-150kDa respectively.

I am a bit doubted that
1. Are all these 2 bands specifying the epitotes of the Nav 1.8?
2. If so, for quantification of the expression of Nav 1.8, Should I take into account of one of the bands only or both of them?

I have tried to consult their technical support but yet to get the reply. Just wonder anyone there has encountered similar problem and may kindly give a help.

Moreover, in my practice I was only able to get the second band (100-150kDa). Dunno if it is a problem of my protocol. Briefly, I used the RIPA buffer for sample preparation followed by an 8% gel SDS-PAGE at 160V and transferred to PDVF membrane at 300mA, 130mins and the marker of 250kDa is successfully transferred to the membrane. The membrane is then probed with 1:1000 Anti-Nav1.8 Ab and a HRP conjugated secondary Ab.

Any help would be appreciated, thanks!
Zlll

P.S. Attached with the datasheet illustration

Attached Thumbnails

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[Zlll]

Recenty, I got a reply from the supplier

Quote

The expected MW of the rat Nav1.8 based only in the amino acid sequence is 220kD. However, as you know, there are often modifications such as glycosylations and/or alternative splicing that can affect the apparent MW of a protein in a Western blot. In addition, in our experience, high MW membrane proteins tend to be very "sensitive" to protein degradation during sample preparation, so it is possible that the lower MW bands are degradation products of the Nav1.8 channel.

so if the band I have was the degraded Nav1.8, does it make sense to use it for quantification?
and...any idea for the improvement of my sample preparation in order to preserve the integrity?

thanks in advance!!!

[bob1]

I would guess that the 100-150 kDa band is a non-specific binding of some sort.  It is quite common for antibodies to detect more than one protein, even if the supposed epitope is not present in the non-specific protein. The fact that the bands are removed by pre-incubation with the epitope peptide shows that the smaller bands may be degradation products.

You can take better care of you proteins by adding protease inhibitors (such as cOmplete from Roche) and phosphatase inhibitors.  You can also make sure that you keep them cold and aliquot so that samples are not undegoing several freeze/thaw steps.  Freezing at -80 is a good idea too for unstable proteins.

The epitope of an antibody is the sequence that the antibody recognises within the protein. Usually this is an 8-15 amino acid sequence, but sometimes antibodies are raised against full length sequence, though the actual epitope in this case is still usually only a short sequence.

In my experience, quantitation from westerns is not very viable technique, unless the quantitation is done only within one gel and not compared to other gels as there are many many variables that with very small changes will result in variations in the results you get.

[Zlll]

bob1, on 27 July 2010 - 04:40 PM, said:

You can take better care of you proteins by adding protease inhibitors (such as cOmplete from Roche) and phosphatase inhibitors.  You can also make sure that you keep them cold and aliquot so that samples are not undegoing several freeze/thaw steps.  Freezing at -80 is a good idea too for unstable proteins.

Thanks for your help!
In my case, actually I have added the protease inhibitors(cOmplete)in the RIPA buffer and snap-freezed my tissue samples at -80 before processing.