Out of curiosity ! I was trying to do western blot for Erythropoietin, as you can see the procedure was unsuccesful but there are some unexpected white bands on the blots. Can anyone tell me what might be causing these bands ?
Thank you :)
Exactly what Karsten Krey said. If your expected band size is where you see the white bands, I would recommend using less antibody.
Hi Megha Nimje,
The white band is not the target protein? If not, the results revealed a inspecific detection, the liek previouly said, you must dilute the antibody, is the antidoby comercial available or in house?
regards
Hi Julie Joseph ,
Expected band should be around the dark band below in the blot, around 30 KDa. I kept the dilution at 1:1000 for the primary antibody, even at higher dilutions i am not getting expected results. Dilution for my secondary antibody was 1:5000.
Thank you
Hi Ferdinando B Freitas ,
No, the white bands are not the target protein, the antibodies are commercial from santacruz. Should i increase dilution for primary or secondary antibody ?
Thank you
Hi Megha Nimje
The central lane is the MW marker?
Make sure that you have the amount of total protein needed to detection. I can see a small dark band under the strong band (center), i guess you will need more protein, and it seems the secondary antibody is to concentrated for a comercial available one (1:5000).
Megha Nimje , uploading a gel picture with the marker sizes and your sample types would help. Do you mean to say that the expected band is the dark one at the bottom of the gel in lane 5 or the slightly dark bands below the white band in lanes 1-4 and 6-9?
If it is the thick black band in lane 5, the protein seems to be highly expressed in lane 5 sample but not in the others. You could try loading more of your other samples so you can get better signals. However, I would look into why these samples don't express the protein. From what I see on the gel, lane 1-4, 6-9 samples seem to be of the same kind while lane 6 sample is something totally different (but not the MW marker). Is there a reason why Epo in these samples could be of a different MW? If not, then the bands in these lanes are non-specific (although you may be seeing some of these non-specific bands in lane 5 too, but with slightly higher MW).
I would recommend testing alternate primary antibodies to identify one which is more specific and which might work better with your cells/samples. I have had to test multiple antibodies for some of the proteins I have worked with.
Ferdinando B Freitas Yes, the central lane is MW marker the two dark bands are of size 80 KDa and 30KDa. I will try again with more protein and diluted secondary.
Thank you so much for taking time out to help me.
Julie Joseph , lane 5 is MW marker, lane 1-4 is protein from one group of animals and lane 6-9 is from another group. Thank you so much for the helpful suggestions.
I agree, I would say this is a consequence of bleaching due to high Ab binding to the target. Possibly you should use lower Ab conc. for detection (during first and/ or second antibody incubation).
Andreas Eisenreich , is it possible that i am not using enough ECL substrate ?
In my opinion you, use enough substarte. I think that blocking ist not optimally working and/or the Ab concentration (first or second) is to high. This ,ay lead to bleaching of the bands.
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