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- Proteins and Peptides
- Secondary antibodies
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If your product does not perform as described on this datasheet, we will refund or replace your product...
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Hola , |
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ANSWER: |
Muchas gracias por tu respuesta. |
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A potential customer would like to detect human Oct4, Sox-2, C-myc in |
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ANSWER: |
Thank you for contacting us. |
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Dear Madam or Sir, I am a bit confused about the epitope used for the production of ab18976 (rabbit polyclonal to OCT4). In the datasheet that came with the antibody it is written, that the immunogen was a "recombinant fragment corresponding to amino acids 1-140 of human Oct4". On your website (see http://www.abcam.com/Oct4-antibody-ab18976.html ) it is written that is was a "recombinant fragment, corresponding to amino acids 1-14 of Human Oct4". It would be important for me to know the exact lenght of the immunogen employed! Thanks a lot! |
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ANSWER: |
Thank you very much for your email.
I apologize for this confusion. Unfortunately I do not know where it comes from. I can however confirm that the immunogen used for this antibody is from amino acid 1 to 14. The sequence is AGHLASDFAFSPC.
I hope this information is helpful. Please do not hesitate to contact us again should you have any other question or concern. |
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DESCRIPTION OF THE PROBLEM Non-specific band
SAMPLE mouse ESC lysate
PRIMARY ANTIBODY Concentration or dilution- 1: 1000 Diluent buffer : 5% skim milk TBST Incubation time : O/N Incubation temperature: 4 degree
DETECTION METHOD ECL
POSITIVE AND NEGATIVE CONTROLS USED Positive control : sample is positive control Negative control : MEF
ANTIBODY STORAGE CONDITIONS -20 degree
SAMPLE PREPARATION Lysis buffer : RIPA Protease inhibitors: add Phosphatase:inhibitors: add Reducing agent: reduced Boiling for 5 min
AMOUNT OF PROTEIN LOADED 25ug
ELECTROPHORESIS/GEL CONDITIONS Percentage of gel: 10% Type of membrane : NC
TRANSFER AND BLOCKING CONDITIONS Protein transfer verified: 4 degree Blocking agent and concentration: 5% skim milk Blocking time : 1hr 30min Blocking temperature: RT
SECONDARY ANTIBODY anti rabbit IgG Concentration or dilution : 1: 2000 Diluent buffer: 5% skim milk TBST Incubation time: 2hr Incubation temperature: RT Fluorochrome or enzyme conjugate: HRP washing Buffer : TBST Number of washes : 3 times
HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes
WHAT STEPS HAVE YOU ALTERED? blocking buffer solution: BSA-> Skim milk
ADDITIONAL NOTES Our user could not detect specific result from your product. Thus he wants to issue a credit note from you. I attached an image file please check this for our user. I look forward to hearing from you. |
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ANSWER: |
Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.
Having reviewed this case, I would like to offer some suggestions to help optimise the results from ab18976. I would suggest the following:
-a no primary control to be performed to check if the non-specificity is as a result of the secondary antibody.
-the incubating time of the primary antibody reduced to 2 hours at RT.
and the addition of a mild detergent such as Tween 20 to the incubation and washing buffer and/or removing the milk from the incubation buffer may also help.
Should the suggestions not improve the results, please do let me know.
In the event that a product is not functioning in the species and applications cited on the product datasheet, as in this case (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement or credit note. |
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The same secondary was tested with beta actin successfully. I'll see about the F9 and picture. Thanks |
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ANSWER: |
Tanya is still unwell and I would like to reply to you to prevent further delays. I have looked at the customer' protocol again and I think it will be very important to run a good positive control along the customer's samples. I was unable to find any published reference for the presence of oct4 in human prostate cancer, the only reference for a similar study was on human testicular cancer where IHC studies showed oct4 present in dysplastic cells of intratubular germ cells (Jones et al, OCT4: A sensitive and specific biomarker for intratubular germ cell neoplasia of the testis.Clin Cancer Res. 2004 Dec 15;10(24):8544-7). As the protein is nuclear it may be necessary to do a nuclear extraction to concentrate the protein. I would also recommend to increase the antibody concentration to 2ug/ml and to incubate the antibody overnight. The non specific bands could be due to too much milk, so I would try 1hr 5%BSA and incubating the antibody in TBST only. We don't recommend using ECl as it is not very sensitive, so please ask the customer to use Ecl+ or pierce super signal detection systems. I hope the above information helps, please let me know if the customer still has problems with the positive control and with those suggestions, Have a nice weekend, |
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