Anti-CD44 antibody [F10-44-2] (ab6124)

Product code: 93574
Inquiry: Our customer is looking for the following : CD44 with the clone H90" . The item we offered against his request is AB93574 which include Clone F10-44-2 not clone 'H90'. Kindly advice which Item from ABCAM matches his request. Thank you

ANSWER:

Thank you for contacting us.

Unfortunately, we do not have an anti-CD44 antibody from the H90 clone in our catalogue. We do however have several other antibodies against this target which may be suitable for your customer. The catalogue number you mentioned (ab93574) does not match to an anti-CD44 antibody. The clone mouse monoclonal anti-CD44 clone F10-44-2 is available with many different conjugates which can be viewed from the following link:

http://www.abcam.com/index.html?pageconfig=searchresults&search=cd44&pt=1&sk=clonenum&sv=F10-44-2&sn=F10-44-2&l=3&fViewMore=1

The suitability of this clone will depend on the application and samples your customer intends to use. If you could give me a few more details about this I may be able to advise further as to whether this clone would be suitable or if one of our other anti-CD44 antibodies would be more appropriate.

I hope this information has been of some help. If you would like any further assistance please do let me know.

Zu dem angesprochenen Antikörper „Anti-CD44 antibody [F10-44-2] (PE/Cy7 ®) (ab46793)“ hätte ich noch folgende Fragen.

1.       Bedeutet die Tatsache, dass der Antikörper nicht in  der Liste mit nicht-maus-kreuzreaktiven Antikörpern auftaucht, dass er nicht getestet wurde oder das er getestet wurde und kreuzreaktiv ist?

2.       Der Antikörper ist für die Immunhistologie ausgeschrieben und mich würde interessieren mit welcher Methodik Cy7® detektiert werden kann, da ich es bisher nur aus der Durchflusszytometrie kenne. Zeiss zum Beispiel gibt an kein passendes Filtersystem für Cy7® zu haben.

Ich würde mich über eine Antwort freuen.

ANSWER:

Vielen Dank für die Anfrage.

Dass ab46793 nicht auf der Liste auftaucht, bedeutet dass er nicht getestet wurde, ob er mit Maus kreuzreagiert.

In der Tat ist ab46793 für die Immunhistochemie (IHC-P/Fr - neben anderen Anwendungen wie Durchflusszytometrie) garantiert. Allerdings ist dieser CD44- Antikörper mit dem Tandemkonjugat PE-Cy7 gekoppelt. Welche Anregungswellenlängen beziehungsweise Filter Du für die Detektion benötigst, kannst Du anhand des Spektrums ersehen (Details dazu in Spectra-Viewern von eBioscience http://www.ebioscience.com/resources/fluorplan-spectra-viewer.htm  oder Biolegend http://www.biolegend.com/spectraanalyzer etc.) und beim Hersteller des Mikroskops erfragen. Im Allgemeinen sind PE und Tandem-Fluorochrome wie PE-Cy7 jedoch recht instabil und besitzen eine schwache Fluoreszenz-Intensität, so dass sie in der Regel wenig für die Mikroskopie geeignet sind. Wie auf dem Datenblatt von ab46793 beschrieben, würde ich deswegen für die Detektion dieses CD44- Antikörpers in der IHC einen sekundären Antikörper wie zum Beispiel ab96879 oder ab98697 empfehlen (je nach Fluorochrom-Belegung bei einer Mehrfachfärbung beziehungsweise vorhandenem Filtersystem). Daher ist es eigentlich nicht notwendig, den Klon [F10-44-2] als PE-Cy7-Konjugat zu nehmen (es sei denn, es sind noch andere Experimente damit geplant), sondern Du kannst ihn auch unkonjugiert erwerben (ab6124):

ab6124: Click here (or use the following: http://www.abcam.com/index.html?datasheet=6124). ab96879: Click here (or use the following: http://www.abcam.com/index.html?datasheet=96879). ab98697: Click here (or use the following: http://www.abcam.com/index.html?datasheet=98697).

Ich hoffe, diese Informationen helfen Dir weiter. Bitte zögere nicht, Dich wieder bei uns zu melden, falls Du weitere Fragen hast. 

Here you are my answers to abcam in details regarding my project so please forward to them and hopfuly I can get their guide asap. I am isolating human hertwig's epithelial root sheath (HERS) cells from periodontal ligament (PDL). So the cells that I am isolating are epithelial that should express these two epithelial markers as published in the literature (E-cadherin and Pan-cytokeratin) and will compare them with Stem cells from Human exfoliated deciduous teeth (SHED) using another two anibodies (CD44) that should be highly expressed in SHED than HERS and (CD73) that will be expressed similarly in both cells. So for characterization I ordered these antibodies from abcam: 1-      Mouse monoclonal (HECD-1) to E Cadherin (ab1416) 2-      Mouse monoclonal (PCK-26) to pan Cytokeratin (ab6401) 3-      Mouse monoclonal (F10-44-2) to CD44 (ab6124) 4-      Mouse monoclonal (7G2) to CD73 (ab54217) -          Immunofluorescence staining steps (first time) HERS were cultured in 4 well culture slide and the second day we did the following: a-      Discard the medium b-     Wash 3 times with PBS each time two mins c-      Add 500 µL methanol to each well for 20 mins at 4°C d-     Discard methanol and wash 3 times with PBS each time two mins e-      Add 7 drops of blocking reagent (from Millipore/ comes in the kit of immunoperoxidase secondary detection system) keep it at room temperature for 1 hr. f-       Discard blocking reagent and add the primary antibodies (1:50 ratio) (each antibody was applied separately in each well of the same chamber slide) (we diluted the antibodies using PBS) g-      Keep the chamber slide in 4°C for overnight. h-     Then discard the antibodies, wash 3 times with PBS each time two mins i-        Add 500 µL of secondary antibody (anti mouse FITC) to each well and keep it for 1 hr at room temperature j-       Discard secondary antibody and wash 3 times with PBS each time two mins k-     Add 500 µL of DAPI to each well keep it for 1 hr in a dark room l-        Discard DAPI and wash properly with PBS m-   The cover of the chamber slide was chopped and fluorescence mounting medium was applied (from Dako: S302380) n-     Cover slip was placed and the observed under fluorescence microscope image analyzer Under microscope: We observed the following (as I sent you previously some images); Green cells with green background and blues nucleus. So how to get black background with only green cells and blue nucleus as we got used for immunofluoescence images? -          We did optimize the method (second time) using the same previous procedures but with primary antibodies 1:300 but we got the same result. -          We did optimize the method (third time) using the same previous steps but with these changes: a-      We used PBS with Tween-20 for proper washing b-     Apply methanol and keep it in -20°C for 5 mins c-      Primary antibodies were applied with ratio (1:500) and keep it for 9 hrs at 4°C d-     DAPI was applied for half hr in a dark room. -          But under microscope we got the same result. So we are planning to use primary antibodies 1:1000 and keep it for 3 hrs but we do really need your recommendation and help to get the correct result before we run the test. And waiting for your new quotation please as I did email you. Best regards,    

ANSWER:

Thank you for contacting us.

The background should be black. I am sorry we never had a question like this before. It looks like this problem might be due to instrumentation then antibodies itself.

I have following recommendations that might help; - Use less number of cells per well. - Check the viability and passage level of cells. - The cells must be healthy with low number of passages. - Avoid drying out plate between washings. - Check microscope settings. - Check if the blocking is sufficiently done. You can also use 3-5% BSA for 1-2 hour in PBST for blocking. - Use more diluted primary antibodies.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.  

Good morning, My Safety department requires that I file MSDS's for the antibodies before I can order them. Can you please provide me with the MSDS sheets for the following antibodies: ab762 (CD34) ab6124 (CD44) ab20147 (CD90) ab11414 (CD105) ab 3125 (p75 NFG Receptor) ab7500 (human IgG)

I already have the MSDS for sodium azide. I need the MSDS for the antibodies only. Please email (clee47@dpyus.jnj.com) or fax them to me at (508) 828-3731. Please call me at (508) 880-8399 if you have any questions regarding this request. Thank you very much. Best regards,

ANSWER:

Thank you for your enquiry.

We do not have MSDSs specific for each antiserum. The material safety datasheets are issued based on the inclusion of additional components such as Na Azide and Glycerol in addition to specific componds that we sell for particular techniques such as DAB for IHC etc.

I hope this makes sense.

Customer's question:

could you tell me where the epitope of this antibody lies (ectodomain, intracellular?) Thanks!

ANSWER:

We regret that we have not determined the epitope specificity for this antibody. However, our data from immunohistochemistry and flow cytometry suggest that the epitope is expressed on the surface of CD44-positive cells.