I just did my first western blot a few weeks ago, and i have been trying to troubleshoot ever since. The problem with my blot is that too many bands are seen on the blot and none of them are for my protein of interest the size i was looking for is 42 kDa. I am not sure what the problem could be? not sure if it is my primary antibody that i am using which is monoclonal anti actin in mouse from sigma (clone AC-40), or if its the secondary antibody which is Peroxidase conjugated affinipure goat anti mouse igG form jackson immuno research. If someone could please help me trouble shoot this issue.......
Ok, so to begin I was using a TGS running buffer to run the gel for about an hr at 100v, i am transferring the proteins on a pvdf membrane and I was blocking the membrane for an hour using 5% non fat dry milk in TBS-T for an hour, after the blocking i was directly putting it in at a 1:500 primary antibody incubation overnight at 4C with gentle rocking. In the morning i would wash it 3X for 5min with TBS-T, and then proceeded to incubate it with the secondary antibody at a 1:5000 concentration and left it for an hour with gentle rocking. After that i would wash it again 3x with TBS-T for 5 min. After that i would use the Super Signal West Femto Maximum Sensitivity Substrate used a concentration of 1mL total and put it on the membrane incubate for 5 minutes and proceed to image.
the first time i used vimentin as an antibody and then stripped blot using abcam mild stripping method, and probed for actin both times still getting alot of of non specific binding.
The second time i tried running the blot used the same thing for the gel TGS running buffer the same amount of time and transferred it to a PVDF membrane, the difference here was that i used BSA fraction V for the blocking instead of the 5% non-fat milk, I did a 10% BSA solution in PBS-T i left the membrane in the blocking buffer overnight at room temp using gentle rocking. the next day i washed the membrane 3x for 5 minutes in PBS-t and then proceeded to do the primary incubation with Actin at a 1:500, and 1:1000 concentration in PBS-T with 0.5% milk added for an 1hr:20min with gentle rocking,then i washed the membrane again 3x for 5min, and proceeded to the secondary antibody incubation at a concentration of 1:5000 in PBS-t for an hour again gentle rocking, then i proceeded to do the washing again 3x for 5 min and put the ECL substrate once again and incubated 5 min before i imaged, by the way i am using the bio-rad chemi doc to image.
Hope that was a bit more detailed, i greatly appreciate the help file:///C:\Users\ELIZAB~1\AppData\Local\Temp\msohtmlclip1\01\clip_image001.png
how to get rid of all the non specific binding siteswestern antibodies
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