Anti-NUMB antibody (ab4147)

Dear Tech support

Could you pleasespeculate regarding ab4147 reactivity against Drosophila derived samples?

Would you like to offer a participation in the abreview promotion for Drosophila with one of the Numb antibodies available.


Thank you for your kind help.

Regards

ANSWER:

Thank you for contacting us.


The anti-NUMB antibody (ab4147) is raised against a peptide corresponding to residues 638-651 of human NUMB. Through sequence alignment of the Drosophila melanogaster protein (SwissProt reference http://www.uniprot.org/uniprot/P16554), this region shares very little homology with the human protein. I would therefore not suggest usign this antibody to detect the Drosophila protein.


Unfortunately we have no other anti-NUMB antibodies that I can suggest as an alternative. You may have more luck by searching Biocompare:


http://www.biocomplare.com


I am sorry that I have not been able to be of more help in this instance. If you have any further questions please do not hesitate to contact us again.

BATCH NUMBER 141307 ORDER NUMBER 96008

DESCRIPTION OF THE PROBLEM Wrong band size: band size is supposed to be approx 71kDa, but I am getting a very robust band at about 115kDa that is much stronger than the band I see at about 70kDa when looking at various mouse cell lysates (see attached file, ab4147 gel on left). When I probe same blot with another Numb antibody (see gel on right), I get much more specificity towards the 70kDa bands. Though I still see a faint band at a larger molecular weight, this is greatly reduced and rather negligible when compared to the robustness of the 70kDa band. Contrarily, the abcam antobody shows the STRONGEST sensitivity to the 115kDa band. Thus, it appears as if the abcam is picking up a non-specific protein in mouse cells (as I mentioned, an abcam technician told me that abcam had only tested this antibody for Westerns on human cell lines), and that this abnormal band is not a product of an abnormal gel or a high level of modification to Numb, as an alternative Numb antibody does not pick up this product. Note: two bands seen at approx 70kDa size are due to four isoforms (65, 66, 71 and 72kDa expacted band sizes).

SAMPLE For both gels, lanes are as follows: 1 - E15 forebrain negative control 2 - 3T3 cell line (mouse fibroblasts) negative control 3 - MHP36 cell line (mouse hippocampal stem cell line) negative control 4 - E15 positive 5 - 3T3 positive 6 - MHP36 positive

PRIMARY ANTIBODY ab4147 used at 1:200 in 5% nonfat milk/TBST, 2 hrs shaking at RT, followed by 4 x 5 min wash in TBST..

DETECTION METHOD ECL - Lumilight Kit

POSITIVE AND NEGATIVE CONTROLS USED E15 - as positive control Lanes 1-3 as negative controls (no primary antibody added)

ANTIBODY STORAGE CONDITIONS Aliquotted and stored at -20oC

SAMPLE PREPARATION Cells lysed in lysis buffer (Tris pH7.5 50mM, NaCl 150mM, Triton X-100 1%, EDTA 5mM) for 30 min on ice, spun and then supernatant isolated. Mixed with sample buffer in 3:4 ratio, incubated ar 90oC for 6 min and loaded into gel.

AMOUNT OF PROTEIN LOADED E15 - 5ul 3T3 - 20ul MHP36 - 10ul

ELECTROPHORESIS/GEL CONDITIONS 8% SDS gel

TRANSFER AND BLOCKING CONDITIONS Gel transfered for 2.5 hrs at 10V. Block 1 hr in 5% nonfat milk/TBST shaking at RT.

SECONDARY ANTIBODY Secondary - Donkey anti-goat IgG HRP, [another company], used 1:2000, 1hr at RT shaking, followed by 4 x 5 min wash in TBST.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Have also tried on 10% SDS gel and with incubating primary overnight at 4oC with same results.

ADDITIONAL NOTES Also ordered same antibody on purchase order number 90419 (have ordered two vials total).

Have spoken with Hugh Spotswood with regards to this matter. As this antibody was advertised as being specific for mouse and does not appear to be, I would greatly appreciate a reimbursement for the two purchases of this antibody. Thank you.

ANSWER:

Thank you for your enquiry and for providing me with details of the western blotting experiments that you have performed.

From the internal notes that I have I can see that mouse reactivity by this antibody was added as a result of two independent Abreviews. Customer feedback following reviews of how the antibody behaves by IP and IHC on formalin-fixed paraffin-embedded sections resulted in mouse being added to the antibody's datasheet. I have updated our datasheet to include the information that you have provided me with.

I am prepared to offer you credit against the purchase of this antibody given that it has not behaved as detailed on our datasheet. This is provided that the purchase was made within the past 90 days. If this is the case please e-mail me details of the order and date of purchase and I will ask our accounts team to raise credit against your initial purchase.

Is the Western blotting protocol that was used to test this antibody available?

ANSWER:

Below is the Western blot protocol that the originator used when characterizing this antibody.

I hope this is helpful. If you need additional assistance, please don't hesitate to contact us again.

- Lysis. Cell pellets were washed with ice-cold PBS. 1 ml of RIPA buffer was added per 1E8 cells and incubated on ice for 20 min, vortexing 2-3 times, briefly. The lysate was aliquotted into 1.5 ml microfuge tubes and centrifuged at 13,000 rpm for 5 min in a microfuge. The supernatant was transferred into clean tubes and its protein concentration was measured with BioRad protein assay. The concentration was then adjusted to 5 mg/ml with RIPA lysis buffer. An equal volume of 2 x SDS sample buffer was then added and the cell lysate was boiled for 5 minutes. Lysates were stored at -80C until use.(RIPA buffer = 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 µg/ml Aprotinin, 5 µg/ml Leupeptin, 1% Triton X-100, 1% Sodium deoxycholate, 0.1% SDS).

- SDS PAGE. Samples were run at 200V constant on a 12% acrylamide SDS-PAGE mini gel - using Biorad Mini-Protean 3 kit and protocols. Before loading samples had 5% (v/v) 2-ME added and were boiled for 3 minutes.

- Transfer. We used a Biorad Mini Trans-Blot, constant 100 V for 1 hour. Transfer Buffer was 20 mM Tris pH 8.0, 150 mM Glycine, 10% Methanol. We transferred to Millipore PVDF membrane and stained with Ponceau Red to evaluate the transfer.

- Staining. The membrane was blocked in 2.5% skimmed milk in TBS-T (TBS + 0.05% Tween-20) for 1 hr at room temperature with agitation. Primary antibody was incubated for 1 hr at room temperature with agitation. We used sigma secondary (Sigma anti-goat-HRP Product # A4174, use 1:3000) for 1 hr at room temperature with agitation. We washed with TBST three times after primary and secondary antibody, each wash lasting for 5-10 mins. ECL-plus (Amersham) was used rather than ECL, which is considerably more sensitive. Final detection was on autoradiography film.

Hello, I already got a reply about a query regarding Numb protein, you said you have no information on where to get this protein. I was wondering how you test your numb antibody (Ab4147) if you don't have the numb protein?

ANSWER:

Thank you for your question. We source this antibody from a particular originator and hence we do not currently have access to the numb protein ourselves. If you would like a positive control for this antibody I recommend you try ab7909 (A431 whole cell lysate). Sorry for the inconvenience,

Succinctly, there is no staining. The level of staining is equal to that of control sections where no primary antibody was applied to the tissue, just blocking solution and secondary antibody. I will merely answer the remainder of your questions as they are asked to make things clear. I am still looking for the lot number; someone labeled over the original label and it is proving difficult to get this number.

2) the tissue is human, fetal central nervous system tissue. More specifically, the tissue ranges in gestational ages from 10 weeks to 24 weeks. Primarily, we are staining cortex only and we are not interested in any subcortical structures.

3)All of the tissue was fixed in 4% paraformaldahyde

4)we tried IHC with and without antigen retrieval. Antigen retrieval was tried with heat mediated high ph sodium citrate

5)we use blocking solution containing 0.25% TX and 10% NGS and bovine serum albumin at 20 mg/ 2mL in PBS

6) primary antibody used was ab4147, anti-NUMB, raised against goat. We have tried a range of dilutions from 1:1000 to 1:10 (1:1000, 1:500, 1:200, 1:100, 1:50, 1:20, 1:10) - sections were dried and then rinsed 3 times for 5 minutes in ice cold PBS. Then the sections were blocked for 1 hour (blocking solution is described above). Primary antibody was then aplied and left to incubate at either room temperature of 4 degrees C over night. Sections were then rinsed 3 times for 5 minutes each with ice cold PBS. Secondary antibody was applied for either 1 hour or 2 hours. sections were then rinsed three more times for 5 minutes each with ice cold PBS and then mounted.

7)secondary antibody is molecular probes alexa fluor 555 goat anti rabbit IgG (H+L). Wash steps and incubation are aforementioned. this antibody is generally used (depending on the primary antibody and the tissue) by our lab between 1:500 and 1:2000 diluted in PBS. This is a superb secondary antibody that works with all primary antibodies with which I have tried it. Detection is usually very specific with little background. I would say that the problem we are experiencing is not caused by the secondary antibody. We use fluorescent microscopy for detection.

8) i am currently working on getting images to send you. I should have them by to you by tomorrow morning.

9) as aforementioned, we use fluorescent microscopy

10)positive controls are not applicable to our current research interests associated with NUMB antibody. As a negative control we visualized serial sections using identical protocol omitting application of the primary antibody. In place of the primary antibody, sections were left overnight in blocking solution. Secondary antibody was applied at the appropriate dilution. Experimental sections and control sections look very similar. There is no "high" background to speak of, just a lack of staining.

11)we have tried IHC on human, fetal paraformaldahyde fixed frozen sections and paraffin embedded sections of varying gestational ages. The number of attempts is approaching 25. We have changed the amount of NGS in the blocking solution. We have also tried antigen retrieval. We have varied the concentrations of primary and secondary antibody. We have visualized the sections of different fluorescent scopes.

ANSWER:

Thank you very much for the details that you have provided. One thing that I noticed, the secondary antibody: "goat anti rabbit IgG." Ab4147 was rasied in goat so you need a secondary to goat IgG, such as rabbit anti goat IgG (rabbit polyclonal to goat IgG). Please confirm what secondary you are using.