Anti-Notch1 antibody - ChIP Grade (ab27526)

1) Abcam product code  ab8925                         2) Abcam order reference number or product batch number GR25659-1   3) Description of the problem NICD (dots in nuclei) not detectable, only perinuclear staining   4) Sample preparation: Species                        Mouse Type of sample: Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed paraffin embedded sections, cells in culture, other: fixed frozen sections Sample preparation 1% PFA perfusion plus 0.5% post-fixation Positive control same section stained with another batch (lot nr.911660) of activated Notch1 antibody Negative control only secondary antibody   5) Fixation step Yes/No YES If yes:  Fixative agent and concentration Paraformaldehyde (PFA) 1% Fixation time perfusion for 4 minutes PFA 1%+ 2 hours 0.5% PFA as post-fixation Fixation temperature perfusion at 37°C and post-fixation at room temperature   6) Antigen retrieval method   7) Permeabilization method: Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers? Permeabilizing agent and concentration: PBS 0.3% TRITON X-100   8) Blocking agent (eg BSA, serum…): Concentration serum 2% + 1% BSA in PBS 0.3% TRITON X-100 Blocking time     1 hour                                    Blocking temperature room temperature   9) Endogenous peroxidases blocked? NO Endogenous biotins blocked? NO   10) Primary antibody (If more than one was used, describe in “additional notes”) : Concentration or dilution 1:50 Diluent buffer  donkey serum 2% + 1% BSA in PBS 0.3% TRITON X-100 Incubation time 1 hour room temperature   11)  Secondary antibody:           Species: chicken Reacts against: rabbit Concentration or dilution 1:200 Diluent buffer  donkey serum 2% + 1% BSA in PBS 0.3% TRITON X-100 Incubation time 1 hour room temperature Fluorochrome or enzyme conjugate alexa fluor 647   12) Washing after primary and secondary antibodies: Buffer donkey serum 2% + 1% BSA in PBS 0.3% TRITON X-100 Number of washes 3   13) Detection method IF   14) How many times have you run this staining? Many times on many different sections Do you obtain the same results every time?  No, results depend on batch nr of antibody What steps have you altered to try and optimize the use of this antibody? We tried to adjust dilution     Additional Notes: Other primary antibodies used in the same staining were: -          Rat monoclonal anti CD144 (1:100) detected with a donkey anti-rat 488 (1:200) -          Goat polyclonal anti Dll4 (1:100) detected with a donkey anti-goat 564(1:200) -          DAPI 1x added as a secondary antibody   In attachment two images with two different batches (GR25659-1 bad staining; 911660 good staining with clear intranuclear dots in several cells)   Thank you very much   Best regards,

ANSWER:

Thank you for contacting Abcam Technical Team and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this product. The protocol looks absolutely fine to me and I understand that you had good results previously with different batches. I would like to reassure you that our Abpromise applies to your complaint since you purchased this product within the guarantee period. This means that in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased. Currently, we do not have new batch in stock so I can't offer you a different batch. However, we have some alternative antibodies in our catalogue (ab83253, ab27526), please take a look at them and let me know how you wish to proceed with your enquiry. I look forward to hearing from you soon.

Dear technical team, Our customer would like to purchase 11 antibodies from abcam and needs control for each of antibody. The customer will test IHC-P so, control should be slide type. I have searched only one of them as below and would like to ask check the rest of them.  

ANSWER:

Thank you very much for contacting us - and sending the enquiry as a table. That clear overview was very helpful :-)

As you may know, we have not tested the individual samples for their expression of the mentioned targets. However, according to the literature and other sources such as http://www.proteinatlas.org/ these tissues exhibit high expression of these proteins and thus are suitable controls.

Cat# Product Positive control Slide ab27526 Anti-Notch1 antibody Breast carcinoma ab4697, ab4698 (but also ab4701, ab4700, ab4696) ab7970 Anti-NFkB p65 antibody Spleen (or skin) ab4372 (or ab4365, ab4417)　 ab10800 Anti-Integrin alpha 2 antibody Bladder, gall bladder, kidney　 ab4287, ab4330, ab4347　 ab20142 Anti-Integrin alpha 6 antibody Bladder, gall bladder, oesophagus　 ab4287, ab4330, ab4404　 ab16821 Anti-Integrin alpha V antibody Human breast carcinoma ab4697, ab4698 (but also ab4701, ab4700, ab4696)　 ab52971 Anti-Integrin beta 1 antibody Human breast carcinoma ab4697, ab4698 (but also ab4701, ab4700, ab4696)　 ab75872 Anti-Integrin beta 3 antibody Human spleen lysate and human tonsil tissue. ab4372, ab5146 ab79007 Anti-Integrin alpha 1 + beta 1 antibody Rat gut, heart and lung tissue. ab4620, ab4617　 ab30483 Anti-Integrin alpha 2+beta 1 antibody Bladder, gall bladder,breast carcinoma ab4287, ab4330, ab4697, ab4698 (but also ab4701, ab4700, ab4696)　 ab16822 Anti-Integrin alpha 2b + beta 3 antibody Spleen, kidney　 ab4372, ab4347　 ab78289 Anti-Integrin alpha V beta 3 antibody Bladder, oesophagus　 ab4287, ab4404　

All the above mentioned antibodies are guaranteed to work in immunohistochemistry on paraffin-embedded sections as stated on the datasheet. However, we do not currently have an image of ab10800, ab16821, ab30483, ab16822, and ab78289 in IHC-P. Therefore, I can offer a discount off a future purchase if you buy one of these antibodies now, use it IHC-P and submit an image of your results to us in the form of an Abreview. The discount would be to the value of 1 free primary antibody each. If you decide to go ahead and purchase this product, please contact our Scientific Support team prior to the purchase for more details.

I hope this information is helpful to you. Please do not hesitate to contact me if you need any further advice or information in this regard.

Does ab27526 Notch1 antibody detect active and non active forms of the protein?

ANSWER:

Thank you for your recent telephone enquiry.

On further investigation, I can confirm that the immunogen for ab27526 Notch1 antibody is located at C-terminus and therefore should recognize the cytoplasmic domain, the active form that translocates into the nucleus. It should also recognize the full length Notch 1. In Western blotting, it should recognize both 120 kDa (active form) and 300 kDa (full length).

I hope this is helpful to you. Should you have an further questions, please do not hesitate to contact us.

Does this antibody recognize the activated form of Notch1?

ANSWER:

Thank you for your enquiry.

We have not directly tested this antibody with activated form. However, based on the immunogen sequence, it most likely recognizes activated form.

Should you require any other information, please do not hesitate to contact me.

Could you please let me know the sequence of the immunogenic peptide used for generation of this antibody ? I am interested in checking the cross reactivity with Pig Notch-1.

ANSWER:

The sequence of the peptide used to generate ab27526:

AASGHLGRSFLSGEPSQADV

Please do not hesitate to contact us if you have any other questions.