The band is for a sample of concentrated milk where there should be sufficient alfalactalbumin to be recognized by the antibodies
there can be two reasons. One: there is not enough amount to be detected by coomassie staining on SDS PAGE. but because western detects very low amount of proteins you can see it on western blot. the amount of it present is simply less than the detection limit of coomassie.
two: the alfa lactaalbumin cannot be stained by coomassie which is unlikely. in that case lactalbumin is less than 1% of total protein so it is not detected by coomassie.
try loading more total protein or go for little overexposed silver staining.
You see the alpha lactalbumin band on the transfer membrane after immunodetection but not in the SDS gel after staining it? What kind of staining method do you use? Please keep in mind that in gel staining is usually less sensitive than immunodetection. You could also have a problem with some protease activity that leads to degradation of proteins in your milk sample. Do you see other bands in the SDS gel (e. g., casein)?
Cheers, Christian
there can be two reasons. One: there is not enough amount to be detected by coomassie staining on SDS PAGE. but because western detects very low amount of proteins you can see it on western blot. the amount of it present is simply less than the detection limit of coomassie.
two: the alfa lactaalbumin cannot be stained by coomassie which is unlikely. in that case lactalbumin is less than 1% of total protein so it is not detected by coomassie.
try loading more total protein or go for little overexposed silver staining.
As indicated in the previous answers also, you should take into consideration the sensitivity aspect of immunodetection vs Coomassie Brilliant Blue or Amido Black staining. For getting it straightened, besides increasing the loading amount of concentrated milk, you can also include a positive control such as a lane for recombinant alpha lactalbumin protein. Abnova’s H00003906-Q01 can be an option and it has been shown to stain well with Coomassie Blue http://www.abnova.com/products/products_detail.asp?catalog_id=H00003906-Q01
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