Thank you for taking the time to submit this complaint. We have looked through the protocol that you have forwarded to us, as well as seeking additional information from the originator of the product and seeking the protocol that was used to test this Ab. There are some suggestions that may be made that may help with the results that you are gaining.
1) Incubate the primary Ab for 2 hrs at room temperature and reduce the concentration that you are using the primary Ab at. Fra2 shows a band of approximately 50KDa using this Ab in HeLa cells. This KDa is suspected to be the molecular weight of the phophorylated form of the target, from your description it sounds as though the band of interest is present.
2) From the information that you have forwarded you did not add reducing agents to the gel or buffers. The blot was initially run using gels containing B-mercaptoethanol and SDS on a 8% gel with a 5% attacking gel.
3) Normally I would suggest using additional protease inhibitors leupeptin and pepstatin in addition to PMSF to prevent target breakdown (particularly if you are seeing bands below the expected target size).
4) When initially testing this Ab the wash steps used were 6 changes over the course of one 1hr in PBS/T with vigorous shaking.
5) Is there any non specific binding observed when the no primary control is run?
6) Test Sample Preparation. The cells for testing this Ab were snap frozen before storage, this may be an important step. Total cellular proteins were isolated by suspending the cells in 10 vol of 1x Laemmli sample buffer (0.062 M Tris/HCl, pH 6.8, 5% glycerol, 2% SDS) and then sonicating the cell lysate on ice. Protein was estimated using a Bio-Rad DC Protein Assay Kit. Lysates were flash frozen in liquid nitrogen and stored at –80 °C.
7) When testing this Ab HeLa cells were stimulated with 1x10-7 M TPA stimulated (2h). You could also run your non stimulated samples against samples stimulated with TPA. The originator of the product states that they received one clean band in HeLa cells.
If you are still experiencing trouble then please get back in touch with me. I have cut and pasted the protocol used to test this product below.
"Test Sample Preparation: Total cellular proteins were isolated by suspending the cells in 10 vol of 1x Laemmli sample buffer (0.062 M Tris/HCl, pH 6.8, 5% glycerol, 2% SDS) and then sonicating the cell lysate on ice. Protein was estimated using a Bio-Rad DC Protein Assay Kit. Lysates were flash frozen in liquid nitrogen and stored at –80 °C.
Gel Conditions: ß-Mercaptoethanol and bromophenol blue tracking dye was added to a final concentration of 1% and 5 ug/ml, respectively. Total cellular protein (20 ug/lane) was separated by discontinuous SDS PAGE (mini gels) using an 8% separating gel and a 5% stacking gel.
Transfer Conditions: Proteins were transferred to nitrocellulose (BA 85, S & S) using a Gelman semidry transfer unit (Transfer buffer: Tris-base 48 mM, glycine-HCl 39 mM, SDS 1.3 mM, methanol 20%, pH 9.2) and stained with Ponceau S to confirm uniformity of transfer.
Blocking Solution & Duration: 5% non fat dry milk and 0.02% merthiolate in PBS (phosphate-buffered saline), overnight at 4 °C, gentle shaking.
Primary Antibody Incubation Time & Temperature: 2 h at room temperature.
Wash Solution Composition, Repetitions & Time: Washed in PBS/T, 6 changes over 1 h, vigorous shaking.
Secondary Antibody: Used HRP-labelled goat anti-rabbit IgG (H + L) 1:5000
Secondary Antibody Incubation Time and Temperature: 1 h at room temperature.
Wash Solution Composition, Repetitions & Times: Washed in PBS/T, 6 changes over 1 h, vigorous shaking. Detection system Amersham ECL kit. Used Amersham Hyperfilm-MP.
Molecular weight of band that is seen: Reported apparent molecular weight ~46 kDa. There is no Fra2 specific signal when using NIH 3T3 cell extracts. There appears to be a Fra2 specific band at ~52 kDa when using HeLa cell extracts. This is the only protein detected by this Ab, and due to its slower migration, it could represent a phosphorylated form of Fra2.
Species, Tissues and Cell Lines Tested: 20% Serum stimulated (2h) mouse NIH 3t3 and 1x10-7 M TPA stimulated (2h) human HeLa cells.
Conclusions and Impressions: Supershift analysis 10/20/95 showed that Fra2 was unable to supershift protein/DNA complexes formed when using NIH 3T3 or HeLa cell nuclear extracts and AP-1 sites. There appears to be a Fra2 specific band at ~52 kDa. This is the only protein detected by this Ab and represents a phosphorylated form of Fra2".