Anti-XPC antibody [3.26] (ab6264)

No signal in blots of lysates of cells known to express XPC

ANSWER:

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

As requested, I have issued a free of charge replacement with the order number **** for the alternative we discussed, ab21078.

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.

Lanes on the image of the datasheet ab21078 has no legend

ANSWER:

Merci pour votre appel de hier ainsi que pour votre patience. Nous nous excusons encore une fois pour la légende manquant pour le ab21078. Nous avons mis ceci à jour maintenant. Les puits correspondent tous à des cellules HeLa, puits nr 1 était fait avec l'ab21078, puits nr 2 avec l'ab21082 et puits nr 3 avec le ab6264. Malheureusement nous n'avons pas l'information actuelle par rapport à la dilution utilisé, mais la recommandation d'utiliser ces anticorps en Western blot à dilution initiale de 1:500 à 1:1000. J'espère que ces informations vous sont utiles. N'hésitez pas à nous recontacter si vous avez des autres questions. Je vous souhaite une bonne fin de journée.

I am interested in purchasing a positive control and I am in the process of doing this. Can you please tell me approximately how much protein was loaded on the gel to obtain the band from the Raji cells. Can you also tell me if they have been stimuluated. Thank you

ANSWER:

In general, we recommend loading 20 µg of the Raji cell lysate per lane for a mini gel. The Raji cell lysate was not stimulated. If you have any more questions, please let me know.

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 65605

DESCRIPTION OF THE PROBLEM I am getting a nonspecific band around 50 kDa but I am not getting any bands around 100 kDa or above.

SAMPLE Whole cell lysates from Human cells. These cells are either primary cells or SV40 transformed cell lines.

PRIMARY ANTIBODY XPC (ab6264) diluted 1:500 in 1 X TBS-T Incubated overnight at 4 degrees Washed 3 times for 5 minutes each in TBS-T after primary antibody removed

SECONDARY ANTIBODY For a secondary antibody I used Goat Anti-Mouse IgG, HRP-conjugate Diluated 1:5000 in TBS-T Incubated for 30 minutes at room temp Washed 3 times for 10 minutes after removal of secondary with TBS-T

DETECTION METHOD Dectected using ECL

POSITIVE AND NEGATIVE CONTROLS USED I did not have any Raji, can I purchase a positive control extract? The gel was loaded with whole cell extracts from HCT116 cells, normal fibroblasts, XPC Complemented cells (purchased from ATCC), and XPC cells (neg control). These extracts were collect both pre and post exposure to 20 Jm-2 of UV. I have seen in many papers that XPC expression is enhanced following UV irradiation.

ANTIBODY STORAGE CONDITIONS Antibody aliquoted upon delivery and stored at -20C.

SAMPLE PREPARATION Lysis buffer used to prepare samples contains: 50 mM Tris pH 8, 250 mM NaCL, 1% NP40, 0.1% SDS, 5 mM EDTA, 2 mM Sodium Vanadate, 10 mM Sodium Pyrophosphate, 10 mM Sodium Fluoride, 1 ug/ml Aprotinin, 1 ug/ml Leupeptin, 1 ug/ml Pepstatin and 1 mM PMSF

AMOUNT OF PROTEIN LOADED 50 ug

ELECTROPHORESIS/GEL CONDITIONS Samples were run on a 8% SDS-PAGE gel

TRANSFER AND BLOCKING CONDITIONS Transfered for 1 hr at 100V Blocked in 5% Milk prepared in 1 X TBS-T for 1 hr

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Increased antibody concentration and amount of protein loaded

ADDITIONAL NOTES I have stripped each blot and blotted for tubulin and protein is present I would appreciate any ideas as how I can this antibody to work. You can contact me via e-mail or by phone

ANSWER:

Thank you for your enquiry and I'm sorry to hear that you are experiencing difficulty with this antibody. Yes, ab6264 was tested and characterized using Raji whole cell lysate (there is a Western blot image on the online datasheet for ab6294). It is difficult to say what your results mean with comparing them with a positive control. We do sell the Raji whole cell lysate (it is ab7908), or maybe you can borrow some from another lab. At this point, I do strongly suggest running a positive control. If you have any additional questions, please do let me know.

I am curious to know what E. Coli expression vector and bacterial strain was used to produce the antigen XPC used to elicit the antibody response?

ANSWER:

Thank you for your enquiry and your patience.

The strain that was used was BL21 (DE3). Unfortunately, the vector that was used is proprietary information that we cannot reveal.