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HOME CONSERVATION C-TERMINAL ANALYSES DNA AND RNA SEQUENCES RESEARCH QUESTIONS RESULTS RESOURCES REFERENCES |
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HOME CONSERVATION C-TERMINAL ANALYSES DNA AND RNA SEQUENCES RESEARCH QUESTIONS RESULTS RESOURCES REFERENCES |
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Commercial anti-ZNF292 Antibodies Score Card
Testing of commercially available antibodies to ZNF292 on western blots. We need at least one anti-body that we are confident will bind ZNF292. We have tested anti-bodies on the Jukart cell line (whole cell, ABCAM ab7899, nuclear extract ab14844) and HUVEC cells (ATTC, whole cell, isolated nuclei and nuclear extract). Full-length isoform 1 is expected to be ~305K. Key Findings: Though many of the anti-bodies tried show some reactivity, few bind a protein in the calculated weight range for full length ZNF292 isoform-1 (~305KDa). Those polyclonals against the peptide sequence 2000-2050 show the best results. The 305KDa band never appears in whole cell lysates likely due to a preponderance of competing glycoproteins in that molecular weight range. A strong 305KDa band can be seen with nuclear fractions from HUVEC cells. Though one manufacturer reported using Jukart cells we were not successful at visualizing the 305K band with a Jukart nuclear extract ab14844. We used the p.2000-2050 peptide as an immungen to block antibody binding on western blots demonstrating its specificity. |
| Antibody | Region | Likely x-reactivity † |
HUVEC Whole Cell |
HUVEC Nuclei |
HUVEC NEX | HUVEC ChIP Native Whole Cell |
HUVEC Oligo P Native Whole Cell |
|---|---|---|---|---|---|---|---|
| LS-C151260 | p.398-447 |
RLF ZNF654 |
++~76K | +~90K | |||
| HPA050618 | p.1082-1180 | None | |||||
| PA5-65628 | p.1082-1180 | None | |||||
| LS-C386714 | p.1280-1360 | None | - | ++Multiple | |||
| LS-C742744 | p.1311-1360 | None | - | ++Multiple | |||
| A303-267A | p.2000-2050 | None | - | +++~305K* +Multiple |
+Multiple | ||
| ab117769 | p.2000-2050 | None | - | +++~305K* +Multiple |
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| LS-C289953 |
p.2000-2050 |
None | |||||
| LS-B3099 | p.2081-2097 | Many with .01<E<1 | |||||
| LS-C805526 | Internal | ? | - | +Multiple | |||
| PA5-39542 | Internal | ? | - |
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ZNF292 Antibody Tests Two of seven comercially available antibodies tested bind a protein of the expected molecular weight for isoform 1 of ZNF292 (~305KDa). Test of several anti-ZNF292 rabbit polyclonals on crude nuclear fraction from HUVEC cells. 
Crude (N1) and washed (N2) nuclei preparations from HUVEC cells run on 6% SDS-PAGE with higher-weight molecular markers for more accurate size estimation of putative ZNF292 band. Probed with two commercially available affinity- purified anti-bodies: Bethyl A303-267A and abcam ab117769. 
Antibodies blocked by peptide immunogen. The antibodies Bethyl A303-267A and abcam ab117769 were raised against the ZNF292 isoform 1 amino acid sequence p.2000-2050: SRSTQVKKQLAMTEENKKESQPALELRAETQNTHSNVAVIPEKQLVEKKSP We employed GeneScript to create the peptide. The peptide was incubated with A303-267A for 2hrs prior to western blotting. The peptide effectively blocked binding to the 305KDa band plus a lower molecular weight band. It did not block binding to the lowest weight band (likely non-specific) making it a serendipitous loading control. 
ZNF292 appears to be tightly bound in the nucleus. We performed an additional nuclear extraction step on the crude nuclear fraction. The nuclei were incubated in high salt with protease inhibitors and benzonase. This has the effect of forcing loosely bound proteins out through the nuclear pores. Proteins tightly bound to chromatin would be expected to remain in the nuclei. A serial dilution of the nuclear pellet remaining after the extraction was run next to an equivalent fraction of the nuclear extract. The 305KD ZNF292 band can be seen in the pellet but does not appear in the nuclear extract though some of the lower weight bands can be seen. 
ZNF292 Antibody Tests: negative data Test of several anti-ZNF292 rabbit polyclonals on Jukart whole cell lysates (ab7899) 
Test of several anti-ZNF292 rabbit polyclonals on Jukart whole cell and nuclear extracts. 
Other antibodies to be used Heterochromatin Protein 1 Complex Components. Antibodies to three components of HP-1 were tested on HUVEC whole cell lysates: CBX1 (HP-1 β ab152114), CBX3 (HP-1 γ ab217999) and CBX5 (HP-1 α ab227253). Thus far we've only been able to detect HP-1 γ in HUVEC cells. 
Test of anti-Lamin A/C rabbit polyclonal (PA5-79605), anti-GADPH (ab9485) and CBX3 (HP-1 γ ab217999) on nuclear extract from HUVEC cells (NEX) and remaining pellet (NP). 
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