I use TBS or TBS-T with 5% milk to incubate my membrane with the first antibody. The incubation is overnight in cold room, and sometimes the solution changes overnight and the next day it is not white (like milk) but is almost clear. What is the reason for that? The rest of the milk was o/n in the fridge in a bottle and didn't turn. So it must be due to do antibody? I am using the antiflag M2 antibody from Sigma. Thx for your answers...
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#2
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Posted 01 November 2009 - 02:22 AM
I never had this during western blotting. What type of milk are you using? Has it only just started happening?
Does it effect the outcome of the blotting? If it doesn't then I don't worry about it.
Does it effect the outcome of the blotting? If it doesn't then I don't worry about it.
#3
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Posted 01 November 2009 - 02:59 PM
Does it happen with both TBS and TBS-T?
Milk will go clear when the micelle structure is destroyed. Typically this will be because the Ca has been removed (a handy tip if you need to filter sterilize the milk) but in your case probably the micelle proteins are being dissolved by tween or bound to the membrane.
Milk will go clear when the micelle structure is destroyed. Typically this will be because the Ca has been removed (a handy tip if you need to filter sterilize the milk) but in your case probably the micelle proteins are being dissolved by tween or bound to the membrane.
#4
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Posted 09 November 2009 - 04:48 AM
Superman, on Nov 1 2009, 03:22 AM, said:
I never had this during western blotting. What type of milk are you using? Has it only just started happening?
Does it effect the outcome of the blotting? If it doesn't then I don't worry about it.
Does it effect the outcome of the blotting? If it doesn't then I don't worry about it.
DRT, on Nov 1 2009, 03:59 PM, said:
Does it happen with both TBS and TBS-T?
Milk will go clear when the micelle structure is destroyed. Typically this will be because the Ca has been removed (a handy tip if you need to filter sterilize the milk) but in your case probably the micelle proteins are being dissolved by tween or bound to the membrane.
Milk will go clear when the micelle structure is destroyed. Typically this will be because the Ca has been removed (a handy tip if you need to filter sterilize the milk) but in your case probably the micelle proteins are being dissolved by tween or bound to the membrane.
Thank you people. I use TBS, and milk powder. I prepare a solution freshly and incubate with different antibodies o/n. The next morning the milk turns clear however only with some antibodies, whereas with other antibodies it does not turn. I guess it is due to the antibody (Sigma anti-flag, M2), because the same milk does not turn with another antibody.
#5
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Posted 18 November 2009 - 05:37 PM
username, on Nov 9 2009, 04:48 AM, said:
Superman, on Nov 1 2009, 03:22 AM, said:
I never had this during western blotting. What type of milk are you using? Has it only just started happening?
Does it effect the outcome of the blotting? If it doesn't then I don't worry about it.
Does it effect the outcome of the blotting? If it doesn't then I don't worry about it.
DRT, on Nov 1 2009, 03:59 PM, said:
Does it happen with both TBS and TBS-T?
Milk will go clear when the micelle structure is destroyed. Typically this will be because the Ca has been removed (a handy tip if you need to filter sterilize the milk) but in your case probably the micelle proteins are being dissolved by tween or bound to the membrane.
Milk will go clear when the micelle structure is destroyed. Typically this will be because the Ca has been removed (a handy tip if you need to filter sterilize the milk) but in your case probably the micelle proteins are being dissolved by tween or bound to the membrane.
Thank you people. I use TBS, and milk powder. I prepare a solution freshly and incubate with different antibodies o/n. The next morning the milk turns clear however only with some antibodies, whereas with other antibodies it does not turn. I guess it is due to the antibody (Sigma anti-flag, M2), because the same milk does not turn with another antibody.
May your antibody contain some chelators like EDTA or EGTA?
May it be that you use different containers for different antobodies?
