white sample band but black positive control band

[wilairat]

Hi
I have some western blot’s problem ,I expose films in western blot’s protocol .There are white sample band on film. But the marker ,positive control and non specific band are black band. I used primary antibody dilution is 1:1000 and secondary dilution is 1:5000
My samples are tissue’s rat. I load sample volume 20 µg
Primary antibody is antiPV and secondary antibody are anti mouse +antibiotin
I don't know to clear this problem,please to tell me .thank you

[danfive]

Hi,

Seems to me that your western blot is basically positive but that gel migration is a problem.
Try reducing the amount of total protein loaded.
And if native protein conditions are not needed, you may need to add more reducing agent (like BME) and extend heating time (95C for 5-6min).
This is obvious but I'll mention it anyway, you need a balance between the protein and reducing agent or else you'll have a mix of folded, unfolded proteins---which can lead to multiple positive bands.
BTW---those white bands (I get them too) they show up because a lot of non-target protein is present at that size. You may want to simplify the lysate by filtering through a MW cutoff filter. Although in your case because those positive bands are so strong (in samples) you can afford to just load less total protein, and improve the electrophoresis.

[wilairat]

Thank you for your advise
I use BME and sample buffer mix together dilution is 1:19 and I top up it more than sample 5 folds.should I increase BME ????

electroproresis: maybe i have problem too
gel melt : i run it in ice pack and use 10 mA (constant amp.)for 3 hour size protien is 11 kD

[danfive]

Originally Posted by wilairat

Thank you for your advise
I use BME and sample buffer mix together dilution is 1:19 and I top up it more than sample 5 folds.should I increase BME ????

electroproresis: maybe i have problem too
gel melt : i run it in ice pack and use 10 mA (constant amp.)for 3 hour size protien is 11 kD

Start with less protein and a little more BME, also heat the mixed protein and sample buffer for 5 min at 95-100C.

From the picture it looks like there are strong bands in the sample wells---if that is so, you definitely have a problem getting the protein into the gel. Loading less protein should solve that. If not, perhaps a gradient gel or using a lesser concentration gel like going from 12% to 10% Tris-Cl gel may do the trick.

[proatpcr]

I am curious, in your positive control which is your right size protein the top or the bottom one? It seems your bottom one due to the higher signal of that band, however there are 2 higher bands.

If it is your top ones, then you may be ok.

Danfive is correct, you should probably decrease your gel percentage to get better separation of the bands. There seem to be 3 bands in your positive control. Not sure if your protein (positive control) is purified well, or has been degraded to a smaller band (and has a modification as shown as the 2 bands on top).

Interesting.

[admin]

Hello Wilairat,

usually white bands also called GHOST bands are due to ECL problems or antibody problems, however sometimes I don't worry about them much.

If your white bands are a problem, they are usually due to excessive signal generated by your proteins during ECL. You can try reducing antibody or protein concentrations to get rid of your white bands.

The reason you get white bands is because excessive antibody or protein can cause extremely high levels of localized ECL signal (which is seen usually at single bands).

The result of is rapid, and complete consumption of ECL substrate at this point which basically caused no light production after the completion of this reaction, and so when you go to expose your film in the dark room, you only see white bands when your gel is exposed to film.

Pierce has gone as far as writing up a paper on the topic.
Check this:
http://www.piercenet.com/files/AN0011dh5.pdf

Please let us know how you fix this problem (my suggestion is to run a decreased percentage gel and reduce your protein concentration so you can get sharper bands), it is a very interesting question!

Good luck

Cheers

[wilairat]

I am very serious becouse the white bands are band that I need.
I think and try much time to clear this problem.
Thank you for everybody advice
I will decreased percentage gel and reduce my protein concentration.
If I fix this problem I will be happy very much.