Smearing after IP - (Aug/17/2005 )
Hi,
I've been having some problems with smearing on my WBs after IP, which makes it difficult to see and quantify the bands. I only see this smearing pattern in IP samples, not crude extract or supernatant. Can anyone offer any advice?
Some of the explanations I have seen for smearing are:
1) too much protein. However, I have run the supernatant next to the IP'ed samples and don't have the same smearing in the supernatant lanes, although presumably there is more protein in the supernatant compared to IP lanes. I suppose the antibody used for IP increases the amount of protein, but I'm not sure how much I can get away with reducing this by.
2) incorrect pH of buffers: I'm not totally sure what the specific pH's should be...
3) high salt concentration in sample - how can I check this and how do I get around this if this is the case?
I would be very grateful for any advice or ideas. Many thanks. 
Hi
How is your IP so far? Do you think the smearing is due to degraded proteins or antibodies? Maybe the washes should be less vigorous?
W
I've been having some problems with smearing on my WBs after IP, which makes it difficult to see and quantify the bands. I only see this smearing pattern in IP samples, not crude extract or supernatant. Can anyone offer any advice?
Some of the explanations I have seen for smearing are:
1) too much protein. However, I have run the supernatant next to the IP'ed samples and don't have the same smearing in the supernatant lanes, although presumably there is more protein in the supernatant compared to IP lanes. I suppose the antibody used for IP increases the amount of protein, but I'm not sure how much I can get away with reducing this by.
2) incorrect pH of buffers: I'm not totally sure what the specific pH's should be...
3) high salt concentration in sample - how can I check this and how do I get around this if this is the case?
I would be very grateful for any advice or ideas. Many thanks.
Thanks for your reply Winnie. The details of my IP protocol are as follows:
1x Lysis buffer:
10mM Tris HCl, pH 7.6
5mM EDTA (disodium salt)
50mM NaCl
30mM Na pyrophosphate
50mM NaF
1mM Activated Na orthovanadate
1% Triton X-100
Before use add 1:100 dilution of Halt Protease Inhibitor Cocktail (Pierce).
1. Cells are harvested in 1x lysis buffer (above), scraped and transferred into microcentrifuge tubes and incubated on ice for 10mins with frequent vortexing.
2. Insoluble materials are removed by centrifuging @ 13,000rpm for 2mins, 4°C.
3. Supernatant is transferred to fresh tubes for BCA protein assay (Pierce).
4. For IP, 2mg agarose-conjugated primary antibody is incubated with 500mg cell lysate overnight, rocking gently at 4°C.
5. Immunocomplex is washed 3x (centrifuging @ 13,000rpm, 2mins, 4°C and resuspending in 0.5ml lysis buffer).
6. After last wash, 30ml/tube of 2x Laemmli Sample Buffer is added to the beads for denaturing (95°C for 10 mins).
7. The samples are then run on a mini-gel.
I don’t think there should be much protein degradation because the protease inhibitor is present throughout the procedure and for all the washing stages. Do you think that perhaps the denaturing step could be too vigorous?
D