question on pulldown - (Nov/10/2006 )

hello, guys!
GST pulldown puzzled me almost two months! story is like this:
i want to identify interaction between A and B. GST-A and myc-B are used and collected by prokaryotic and eukaryotic expression respectively.
the process is: 1. GST-A is purified with glutathion sepharose beads successfully
2. the beads with GST-A and GST(negative control) were blocked with 0.1 percent BSA 10 hr
3. Then the beads were incubated with myc-B(myc-B is from eukaryotic cell lysate)
4. the pellet was loaded then ditected by myc Ab
negative control is GST-beads
positive control is myc-B cell lysate
negative control and A-GST-beads were quantitized, then the quantity of GST is more than the quantity of GST-A.
my question is whether the wash buffer can affect the interaction, (my wash buffer is 0.1 percent TritonX-100, 60mM NaCl, 10 percent glycerol, 1mM EDTA, 20mM HEPES)
my sample and negative control show the same quantity on autoradiograph

i will go mad!!

---

whooooo give me adviceeeee!!

---

"my sample" is the pelleted pull-down complex of myc-B-A-GST-beads, ok? what is radioactivly labeled?

incubation buffer and washing buffer which should be same or at least similar for the main components and parameters, are indeed critical; the buffer should favor the binding of both proteins; ionic or hydrophobic interactions or a mixture may bind proteins together; so, if there is anything known about which type of binding interaction is realized it should be promoted by the buffers; moreover, unspecific binding to GST-sepharose may happen; I think that is the reason why triton x-100 is added; there may be other reasons for high background and unspecific binding:

low frequency of washing (but I suppose you wash extensively)

or wrong buffer: choose a higher as well as a lower ionic stregth; also, pH can cause trouble, do you know the pI of A and B? try different pH between pH6.5 and pH8.0

---

None is radioactively labeled, the result is shown by mouse anti-myc antibody and HRP conjugated anti-mouse antibody.
the wash buffer and incubation buffer components are same.
the pI of A do not need being checked, because my labmate do interaction between A and C(another protein), the pH7.2 work ,i think pI of B maybe affect the interaction.
how to adjust the the pH between pH6.5 and pH8.0, depending on pI of B.
thanks to kosmodrom

---

I was puzzled about radioactivity as you wrote about autoradiographs but what you meant are autoluminographs;

working with buffers and pI is theoretically simple: using a buffer below your pI of protein X, it carries a positive net charge, above the pI a negative net charge; if you find out the pI for protein A and B, you may find a pH were they carry opposite charges where you may have a good ionic interaction; so, in this case chaotropes should be diminished

---

first of all, i say sorry to kosmodrom, because of my bad expression ,you are puzzled. my way of getting result is by autoluminograph. pI of A is 8.000 and pI of B is 11.0, so as your method ,i should choose pH between 8 and 11. but my labmate do interaction between C(pI 8.000) and D(pI 9.13), he succeed in the identification of interaction at pH 7.4. why? thanks to kosmodrom

---

it´s a question of optimization in trial and error; beside interaction by charge, hydrophobic interaction is possible or as I said before a mixture of charge and hydrophobic interaction; you may not circumvent to find out the right conditions by some more experiments;

moreover, harsh pH may damage or denature your proteins, so one has to be careful

---