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ab46666 |
If your product does not perform as described on this datasheet, we will refund or replace your product...
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Customer reported 12 ul instead of 50 ul in their vial of ab7030. Order# 89977. Lot# 107597. |
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ANSWER: |
Thank you for your phone call and I apologize for the shortage that you have received. I have arranged for a free of charge replacement vial of ab7030 to be sent to you. It is being sent to your attention on order# 93199 and you should receive it Wednesday. If you have any additional questions, please let me know. |
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I have a customer who is using this antibody in cell lysates and tissue lysates. The cell lysates give a single band at ~50 kDa, but the tissue lysates give an extra band at 37 kDa. Although tissue westerns are generally dirtier, and clue or feedback so far on what this band may be?
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ANSWER: |
Thank you for your enquiry. 1. It would be interesting to know what type of cell and tissue lysate the customer used and compared to each other. Were the samples prepared in exactly the same way? Did he/she use RIPA buffer for the lysate? 2. What was the species? 3. Did the customer apply whole cell lysate or nuclear extract? |
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I am interested in doing a IP with your HDAC3 antibody (ab7030), tell me how good is ab7030 in IP assay? Fan
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ANSWER: |
This antibody has not been tested for IP, therefore we cannot guarantee results.
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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"
Sonicated Chromatin prepared from untreated (UI) or 17beta-estradiol (E2) treated MCF7 cells was subjected to the ChIP procedure with ab7030 to HDAC3 and the immunoprecipitated chromatin was analysed in the proximal region of the estrogen-responsive pS2 promoter (as shown above) and quantified by real-time PCR (values are nomalized over inputs). The primers are designed to follow the nucleosome E (including the Estrogen Responsive Element ERE). 2
Sylvain Daujat
Cells were fixed with 4% formaldehyde in PEM buffer. The coverslip was incubated in blocking buffer of 5% powdered milk in TBS-T plus 0.02% sodium azide for 1 hour at room temperature. Blocking buffer was removed and primary antibody was added at a dilution of 1/5000 and incubated overnight at 4 degrees celsius. The coverslips were then washed 4-5 times with blocking buffer for 5 minutes. Secondary antibody, goat anti-rabbit Alexa 594, was added at a dilution of 1/1000 and incubated at room temperature for one hour. From this point on coverslips were covered with foil to protect them from light. They were washed 5 times with TBS-T and then one time with PEM, for 5 minutes each wash. The coverslips were fixed 10-30 minutes in 4% formaldehyde in PEM buffer, then washed 3 times with PEM buffer for 5 minutes. 0.1M ammonium chloride in PEM buffer was added for 10 minutes to quench auto-florescence, and then slips were washed 2 times for 5 minutes in PEM followed by 3 washes for 5 minutes in TBS-T. Coverslips were then counterstained with DAPI in TBS-T for 1-2 minutes, TBS-T was then added and the coverslips mounted. Red indicates staining by ab7030, blue by DAPI. A small amount of cytoplasmic staining was seen with ab7030.
Michael Mancini, Baylor College of Medicine
All lanes : Anti-HDAC3 antibody - ChIP Grade (ab7030) at 1/10000 dilution
Lane 1 : Whole cell lysate from human HEK293 cell line
Lane 2 : Whole cell lysate from human HEK293 cell line treated with HDAC3 gene silencing shRNA
Lysates/proteins at 20 µg per lane.
Secondary
HRP-conjugated goat anti-rabbit Ig at 1/5000 dilution
developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 49 kDa
Exposure time : 10 seconds
This image is courtesy of an anonymous Abreview
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