Products:Epigenetics and Nuclear Signaling >> Transcription >> Domain Families >> Zinc Finger
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We are observing bands in Western blots using ab64457 on various cell lines that are slightly bigger than overexpressed fullength MR or the bands observed from mouse and rat cortex. I would very much like to know the peptide sequence that was used for immunisation, it would help us to decipher what we actually are observing using this antibody. |
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ANSWER: |
Thank you for your inquiry.
Unfortunately, we are not able to release the immunogen sequence for this particular antibody as it is proprietary information. We aim to provide as much information as possible to our customers, so I am sorry that this has not been possible on this occasion.
As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.
Several human Mineralocorticoid Receptor isofomes (4) have been described in the literature. Do you know which one(s) the mouse or rat samples may express?
• Could you please specify what size are the non-specific bands? Are they lower or higher than the expected size (107 kDa)? Could you attach an image (saved as a jpeg file) to your e-mail so that I can have a look at the bands.
• Would you be so kind to inform me how the samples were prepared and what sort of buffer was applied?
• If you wish, I could certainly take a look at the Western blot protocol to see if any tips or recommendations can be made to improve the signal. It would be much appreciated if you could complete the following form (attached as a word document).
Thank you for your understanding and co-operation in this matter. I look forward to hearing from you. |
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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"
All lanes : Anti-Mineralocorticoid Receptor antibody (ab64457) at 1 µg/ml
Lane 1 : Kidney (Mouse) Tissue Lysate
Lane 2 :
Lane 3 :
Lysates/proteins at 10 µg per lane.
Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Performed under reducing conditions.
Predicted band size : 107 kDa
Observed band size : 100 kDa (why is the actual band size different from the predicted?)
Additional bands at : 26 kDa. We are unsure as to the identity of these extra bands.
Exposure time : 30 seconds
The 100 kDa band observed is comparable to the molecular weight seen with other commercially available antibodies to mineralocorticoid receptor.
ICC/IF image of ab64457 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab64457, 1µg/ml) overnight at +4ºC. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) HepG2, Hek293 and MCF7 cells at 1µg/ml, and in 100% methanol fixed (5 min) HeLa, Hek293, HepG2 and MCF7 cells at 1µg/ml.
IHC image of Mineralocorticoid Receptor staining in Human Tonsil FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab64457, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX
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