Im new to Western Blots. Im currently working under the analysis part in Image lab. I watched a couple of videos on how to use it and it says to upload two images (Total Protein Transfer and ECL). When it says total protein transfer, does it mean the image you take before you add the Clarity max reagent after blocking with primary and secondary Antibodies?
Hello Andrea
This is what I understood from your question.
I feel the first image is to be taken when the total protein is transferred to the membrane (PVDF or NC) and stained with Ponceau S.
Ponceau S staining is a reversible staining method used to detect protein bands on the membrane after the transfer process has been completed. This staining generates reddish pink protein bands on a clear background.
The second image you may have to take after the addition of Clarity Max reagent and followed by exposure to X-film OR image the membrane with a digital imager.
Regards.
You can normalise to total protein which means you need something to quantify the total protein on the biorad i.e. coumassie stain of the membrane.
An alternative is to quantify to a loading control like GAPDH, B-tub or Actin which you have probed for with a second antibody incubation.
With the bio rad software you then create the lanes and the bands and then the software can calculate the density of the bands and you can export that to excel
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