Hi everybody! I have no ideas why the bands are white after ECL,so may I got some excellent advices from you guys?
Here is my protocol for western blotting.
I tried to run the protein of FASN, about 250kda.
I used 5% separating gel,and electrophoresis as 100V for 2.5h, and then transfered as 15V for 3h by BIO-RAD semi dry.And I also activate PVDF by rinsing it with 100% methanol first.Concentration of primary antibody is 1:1500,and secondary antibody is 1:1000
So I just doubt wehter the concentration of primary antibody and secondary antibody is right or not.
Waiting for your help,Thank you so much.
Dear Ma,
I agree with Asa's comments. I would like to add that you can also try to reduce the exposure time of the visualization. Look for more solutions on this previous topic of RG.
https://www.researchgate.net/post/White_Bands_in_Western_Blot2
This is overexposure, you can flash expose them for 3 seconds and then develop.
Diluting the antibody also has the advantage of saving money, although by varying the exposures it's easier to try more than one condition per experiment!
Thank you all !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! I diluted the secondary antibody as 1:2000,and it came out great!!! but another problem came out,the bands were a little wide,not slim,is that because of the amount of protein? I just added 10ul each hole.
Thank you again!!!!!!
Probably because too much protein in the well.
Concentration of the sample?
Try to reduce the amount of sample loaded into each well
Yes, "wide" bands can be caused by too much protein in the lane. If you load less, you should get a slimmer and tighter band. However, other factors could also be involved. Your electrophoresis and transfer conditions could affect band morphology - try Coomassie staining a gel or using Ponceau S to visualize transferred proteins on the membrane. If a lot of bands look "blobby" and spread out, there might be an electrophoresis or transfer issue.
Some protein modifications can also affect gel migration and band tightness. If you have multiple modified forms (phosphorylated, glycosylated, etc) migrating close together, and you detect with ECL and film, you'll generally see a larger, fuzzy band and not be able to see the separate bands. This happens because the chemiluminescent signal is being generated in a liquid layer and can diffuse - and also because the emitted photons of light bounce around during film exposure, causing bands to appear wider and fuzzy at the edges. The stronger your band, the more this will affect band morphology and sharpness. A digital detection system (particularly with fluorescence) will give you the sharpest bands and can often discriminate multiple bands that run close together.
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