How to increase Western blot sensitivity? - (Aug/19/2006 )
hello there
i want to know how to increase the sensitivity of western blot?
which step is responsible for getting sometimes dense bands and sometimes faint bands?
i did 3 western for the same samples and each one came with a specific density i mean one was very dense all the bands , one is lighter and the third is so faint in all the bands also?
one of my friends said its the problem of second antibody i am using 1:50000 dilution for one hour shaking but he said make it 3 or 4 hours at RT?
thank u all in advance
one hour is enough to get a good signal.
Maybe you would like to increase the concentration of your antibodies?
Try first with second antibody. try several concentrations of second antibody on your sample, without the first antibody, and try to find the lowest dilution that does not give background.
Then you can do the same with the first antibody.
you can also try different blocking solutions. for some antibodies, it can be better to block in milk, for some other BSA would be better... there is no general rule.
For most of my antibodies, the best is to block in BSA 3%, to incubate firts antibody in 0.3% BSA plus tween20 0.05% and second antibody in milk 1% plus tween 20 0.05%.
however, yesterday I couldn't detect a signal with a new antibody, I will try different blocking solutions (as increasing the concentration only increases the background in this case).
good luck.
i want to know how to increase the sensitivity of western blot?
which step is responsible for getting sometimes dense bands and sometimes faint bands?
i did 3 western for the same samples and each one came with a specific density i mean one was very dense all the bands , one is lighter and the third is so faint in all the bands also?
one of my friends said its the problem of second antibody i am using 1:50000 dilution for one hour shaking but he said make it 3 or 4 hours at RT?
thank u all in advance
hi spanishflower
1. since you are doing a das-elisa i would prefer if you expose your membrane for a longer period to the PRIMARY antibody, preferably overnight with shaking. the secondary antobody is is usually not a problem.
2. if you are using goat-anti-rabbit secondary antibodies, i think the dilution you are using is too high. i use sigma ap-gar conjugate at a dilution of 1:5000.
3. what i dont understand is that your three blots show you reactions of decreasing intensity...thus the first was the best, followed by the second and then the faint third. are you sure that the bcip-nbt (if you are using ap) is stored properly.
4. and what do you mean by "all" the bands. is your primary antibody recognizing many?
all the best
- viv
When we want to increase WB sensitivity, we increase the primary antibody conc., for eg. from 1:5000 to 1:1000. This really helps when u have limited samples or low amounts of protein.
Also we use smaller wells, so the protien is concentrated in a smaller area compared to a larger area.
Ur secondary antibody conc. seems to high. U could try 1:5000. I would incubate the primary at RT for 3-4 hrs instead of secondary.
good luck
thankx everybody really
my first antibody is diluted in 3%BSA TBST 1:1250 overnight 4 degree shaking.
my blocking is BLOCK ACE
and i am using smaller wells already and loading 10 microns each well.
hi again
well my professor insists on increasing the incubation time of second antibody is the way to increase the sensitivity and he said if we use 1:10000 diltuion it will cause high background.
well my professor insists on increasing the incubation time of second antibody is the way to increase the sensitivity and he said if we use 1:10000 diltuion it will cause high background.
well sflo (its a big name you have there)
go ahead and do what he says, but with all due respects to your professor, also do a blot on your own. you can run two gels if there are many samples or one gel and transfer it on one membrane. cut the membrane in half after the transfer, treat one as he says and the other as you want.
if the blocking is done well, there will be no background. i have never used fancy stuff, have always used commercially available milk powder from the grocery store, and there is no background ever. you can try adding it in addition to your block ace. in "your" set, do increase the incubation time with the primary antibody to overnight, and bring the gar-ap conjugate to 1:5000.
- viv
hi there
i did 2 sets today with one gel and one membrane but cut into half and then incubate both in the same first antibody dilution 1:1250 overnight at 4 degree with shaking and for the second antibody i use anti mouse by the way i made 2 sets one with antibody 1:5000 for one hour room temperature shaking but i found this:
No backgroung but multiple bands???? i looked for them and the troubleshooting says may be from too high concentration of second antibody???
the other one still 3 hours left to get the results.
i will let u all know.
you could use a biotinylated 2nd antibody and then use (strept)avidin and biotinylated ap or hrp to increase sensitivity.
thankx mdfenko
the second one strangly enough no detection of any bands except the positive control.
my professor said its not recommended to use 1:5000 dilution of second antibody in ECL detection system?
and i am already using HRP antibody
i am going to do it again , wish me luck.
too depressed.