Anti-RUNX1 / AML1 antibody - ChIP Grade (ab23980)

No siganl in western blot.

ANSWER:

Thank you for contacting Abcam earlier today.

I received your voicemail about the lot number that your colleague was sent. We are out of stock of that particular lot number, but if you have any issues whatsoever with the vial that we are sending you, please let me know and I will be happy to help resolve this issue for you.

Hope you have a nice weekend.

Thanks for your quick response and kind offer. Please see the attached packslip for the previous order. I will definitely test out the Ab with the new lot. Thanks again for all your help.

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

We just bought your product ab23980 for western blotting mouse RUNX1 protein. We used 293T cells transfected with myc-RUNX1 plasmid. While anti-myc can detect the protein well, the anti-RUNX1 antibody (ab23980) gave dark background at dilution of 1:1000 and 1:5000 within 15 sec exposure. The background is too dark to see any band if any.

We used 10% NuPAGE gel to separate the protein lysate , used iblot to transfer protein to PDVF membrane, and 5% milk (biorad) in PBST to block the PDVF membrane. Our 2nd Ab woked well with a different product from your company (abcam 11905).

Do you have any suggestions to lower the background?

ANSWER:

Thank you for bringing this to our attention. I think your protocol with the dilutions you are using should not be giving you high background, especially if the myc western blot demonstrates that the samples are not degraded.

We have a new lot of the antibody which I will be happy to send free-of-charge. Please let me know if you are interested in receiving a vial of the new lot.

1) Abcam product code ab 23980
2) Abcam order reference number or product batch number
3) Description of the problem: no band
4) Sample preparation:
Type of sample (whole cell lysates, fraction, recombinant protein…): nuclues vs cytoplasme
Species : Human samples (patients or cell line)
Lysis buffer : RIPA
Protease inhibitors: kit roche complete + PMSF + AEBSF
Phosphatase inhibitors : No
Reducing agent :b-mercapto
Boiling for ≥5 min? 5 min
Protein loaded ug/lane or cells/lane : 2millions cells
Positive control : ME1
Negative control : No
5) Percentage of gel 12%
Type of membrane nitrocellulose
Protein transfer verifiedSee massruller
Blocking agent and concentration
Blocking time No blocking
Blocking temperature
6) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution 1/1000
Diluent buffer Non fat milk 5%
Incubation time onvernight
Incubation temperature: 4°C
7) Secondary antibody:
Species:
Reacts against: Rabbit
Concentration or dilution 1/20000
Diluent buffer Non fat milk 5%
Incubation time 30 minutes
Incubation temperature: RT
Fluorochrome or enzyme conjugate: HRP
8) Washing after primary and secondary antibodies:
Buffer TPBS
Number of washes 3
9)Detection method West DUra HRP
10) How many times have you run this staining? 3
Do you obtain the same results every time? yes
What steps have you altered to try and optimize the use of this antibody?
Document attachment: Attaching images of your blot is strongly recommended and can greatly speed up our investigation of your problem.

ANSWER:

Merci beaucoup pour ces précisions.

N'hésitez pas à nous contacter de nouveau si vous avez d'autres questions.

problème avec le dernier lot de ab23980 reçu (1071842 GR37372-1).
WB et ChIP

ANSWER:

Suite à mon précédent email, pourriez-vous, s'il vous plait, nous communiquer quelques détails concernant votre protocole en remplissant le questionnaire ci-joint. Ces informations sont très importantes pour nous car elles nous permettent d'investiguer la source du problème rencontré avec cet anticorps et de prendre les dispositions nécessaires pour assurer une bonne qualité de nos produits.

Merci et bon week-end.