Abstract
We have demonstrated that the gel shift assay, a powerful method to study protein · DNA interactions under equilibrium conditions, is both an accurate and precise method to measure the affinity of anti-DNA · DNA immune complexes. One difficulty in performing gel shift assays is disruption of protein · DNA equilibria during the time needed for complexes to enter the gel matrix. However, we have found that highly cross-linked polyacrylamide gels, which are known to form non-restrictive matrices, do not perturb anti-DNA·DNA complexes. Using anti-ssDNA BV04-01 as a model antibody, we find good agreement between the dissociation constants (Kd) measureed in the gel shift assay using a 5.4% polyacrylamide gel cross-linked with 0.6% (bis)acrylamide, and those obtained previously by fluorescence quenching. Because gel shift assays require only nanogram quantities of analyte and can be performed in several hours, it is well suited for a range of anti-DNA binding studies.
| Original language | English |
|---|---|
| Pages (from-to) | 185-190 |
| Number of pages | 6 |
| Journal | Journal of Immunological Methods |
| Volume | 177 |
| Issue number | 1-2 |
| DOIs | |
| State | Published - Dec 28 1994 |
| Externally published | Yes |
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All Science Journal Classification (ASJC) codes
- Biotechnology
- Immunology
Cite this
Application of the gel shift assay to study the affinity and specificity of anti-DNA autoantibodies. / Stevens, Shawn Y.; Swanson, Patrick; Glick, Gary D.
In: Journal of Immunological Methods, Vol. 177, No. 1-2, 28.12.1994, p. 185-190.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Application of the gel shift assay to study the affinity and specificity of anti-DNA autoantibodies
AU - Stevens, Shawn Y.
AU - Swanson, Patrick
AU - Glick, Gary D.
PY - 1994/12/28
Y1 - 1994/12/28
N2 - We have demonstrated that the gel shift assay, a powerful method to study protein · DNA interactions under equilibrium conditions, is both an accurate and precise method to measure the affinity of anti-DNA · DNA immune complexes. One difficulty in performing gel shift assays is disruption of protein · DNA equilibria during the time needed for complexes to enter the gel matrix. However, we have found that highly cross-linked polyacrylamide gels, which are known to form non-restrictive matrices, do not perturb anti-DNA·DNA complexes. Using anti-ssDNA BV04-01 as a model antibody, we find good agreement between the dissociation constants (Kd) measureed in the gel shift assay using a 5.4% polyacrylamide gel cross-linked with 0.6% (bis)acrylamide, and those obtained previously by fluorescence quenching. Because gel shift assays require only nanogram quantities of analyte and can be performed in several hours, it is well suited for a range of anti-DNA binding studies.
AB - We have demonstrated that the gel shift assay, a powerful method to study protein · DNA interactions under equilibrium conditions, is both an accurate and precise method to measure the affinity of anti-DNA · DNA immune complexes. One difficulty in performing gel shift assays is disruption of protein · DNA equilibria during the time needed for complexes to enter the gel matrix. However, we have found that highly cross-linked polyacrylamide gels, which are known to form non-restrictive matrices, do not perturb anti-DNA·DNA complexes. Using anti-ssDNA BV04-01 as a model antibody, we find good agreement between the dissociation constants (Kd) measureed in the gel shift assay using a 5.4% polyacrylamide gel cross-linked with 0.6% (bis)acrylamide, and those obtained previously by fluorescence quenching. Because gel shift assays require only nanogram quantities of analyte and can be performed in several hours, it is well suited for a range of anti-DNA binding studies.
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UR - http://www.scopus.com/inward/citedby.url?scp=0028564754&partnerID=8YFLogxK
U2 - 10.1016/0022-1759(94)90155-4
DO - 10.1016/0022-1759(94)90155-4
M3 - Article
VL - 177
SP - 185
EP - 190
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
SN - 0022-1759
IS - 1-2
ER -