Dear All,
I have Albumin depleted pub plasma samples and I would like to know what conc. can I use for western blotting? Considering the point that my antibody is really specific.
thanks
Best regards
Niharika
Hi, you may run an SDS -PAGE with different concentration of your sample (protein) for example; 1, 2.5, 5 and 10 ug and optimize it with your antibody.
Hi, you may run an SDS -PAGE with different concentration of your sample (protein) for example; 1, 2.5, 5 and 10 ug and optimize it with your antibody.
Niharika: We conjugated HRP directly to all of our primary antibodies and avoided using a secondary anti-IgG-HRP which interact with plasma IgGs. We have run serum (as low as 0.5 microliters) directly for Western blotting and got beautiful band (mostly single!). Good luck.
Hey Niharika,
If I understood it correctly, you are asking the amount of total protein to load on the gel. Then It really depends on the abundance of your protein of interest. Albumin being highly abundant protein and depletion of which gives a better chance to detect low-abundance proteins like cytokines. I would say a short experiment , as suggested by Dr Somasundaram would be a way to know optimal concentration to load with your experimental conditions.
Best Nand.
By the way, it was our experience that most Abs, especially those raised against whole protein plasma Ags, preferentially react with relative "native" form of plasma proteins. Thus, we perform our SDS-PAGE with non-reducing loading buffer (heating is OK) to preserve the disulfide bond.
" preferentially react with relative "native" form of plasma proteins. Thus, we perform our SDS-PAGE with non-reducing loading buffer (heating is OK)..."
Proteins heated in (1% or more?) SDS remain in relatively "native" conformation gives a new meaning to the field of protein structure analysis! ;)
Niharika, in your case, the amount you need to load on gel will entirely depend on:
1. Relative abundance of protein(s) your protein
2. Sensitivity of method of detection
Thank you very much for all great replies. I will keep all of the points in mind.
Thanks again to everyone.
Best regards
Niha
Tausif: "Native" with a quotation mark means that certain proteins can still be active in SDS but without a prior heating treatment. This is the case of a lysosomal enzyme, alpha-N-acetylglucosaminidase, whose activity can be measured in gel slices after a non-reducing SDS-PAGE separation (PMID: 8650226). "(heating is OK)" is a separate sentence to indicate our Western blotting results with no difference in band intensity between heating and non-heating. However, such a reactivity can easily be destroyed upon reducing agent exposure (as low as 2 mM for a brief moment) for most proteins.
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