western blot

Relief to those who perform tedious western blots!!!!

“Biochemists may soon have more time on their hands. Researchers in Michigan have speeded up Western blotting, a biochemistry workhorse for protein separations, by coupling it to an analytical chemistry mainstay, capillary electrophoresis (CE) (Anal. Chem., DOI 10.1021/ac102671n). The new technique produces data in a quarter of the time, and with a fraction of the sample, that Western blots normally use.”

“The result is eliminating the labor-intensive gel-to-membrane transfer.”

Alternative protein-binding scaffolds.

Antibodies are awesome. If you want to find out if a particular protein is in a cell lysate, you look for it using an antibody. Immunoprecipitations and western blots utilise antibodies to identify specific proteins of interest with very high specificity. Antibodies in molecular biology are widespread.

But they’re not without faults. They rely heavily on disulphide bonds for stability, so in a reducing environment, they will come apart. Using harsh conditions for washing or elution can disrupt their binding. Monoclonal antibodies are horrendously expensive to manufacture. Derivatised antibodies containing just Fab or Fv fragments can get over some of these problems, but let’s have a look at the alternatives.

Beta-sandwiches like fibronectin and beta-barrels like lipocalin contain loops which act like the CDR loops, in that they can be derivatised to recognise a specific protein. Methods have been devised so that the phage display of beta-sandwiches, like IgGs, is possible. Affinity is excellent - reported to be nanomolar.

Three-alpha-helical bundles like variants of protein Z recognise proteins via their surfaces. Phage display is possible, and binding is pretty good, down to micromolar affinity.

Repeat proteins like DARPins (designed ankyrin repeat proteins) bind via their repeats, and can be evolved using ribosome display. It’s been reported that this class of scaffold can achieve picomolar affinity of binding!

Active proteins which have intrinsic fluorescence or enzymic activity can do away with the need for enzyme- or fluorophore-linked antibodies, which would normally act as a secondary antibody. These can be evolved using phage display, and the best thing is that you can detect them directly in an ELISA-type assay because they are active.

Currently, the mainstream protein-binder is the antibody. The alternatives are still being developed, and, if they possess desirable properties, are easy and cheap to manufacture, and are not protected by ridiculous intellectual property restrictions, then we might be using them in the lab in some ten years time.

Find more here.

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Blot, blot, western baby. Figure one will be amazing.

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I got a new toy in the lab, irreversible biotin, lovely stuff, can’t wait to get going again, I just hate that you only get 50 micrograms in a pack, which is basically about as much as a pinch of salt, probably less.
My westerns have come on in leaps and bounds, look how nice the top ones are! The bottom one is such a strong signal it just overexposes though, which is a shame.
I have accidentally glutened I think. I don’t know how, but I think I have a lump of cement in my stomach, I can’t concentrate, and I feel sick, such fun.
I think it was the ketchup, everything else was definitely safe.
29th June 2015