Anti-EGF antibody (ab9695)

IHC-P
mouse tumor tissue (from lung) with human cell line injected, some muscle tissue attached as well
sees high background for human cellswith priamry Ab as well as isotype ctrl, but also for muscle (does not contain any EGF)
Ab: 1 ug/ml 3h RT
block: horse serum + chicken egg white albumin
AR: none + proteinase K
2nd (ab96879)

suggested: try no primary crtl to determine if problem is secondary

ANSWER:

Thank you for contacting us.
I am sorry to hear that the antibodies are not working as expected.
As we discussed over the phone, if you please could try the no primary control, this would help us a lot in determining whether the problem comes from the secondary antibody alone, or if there are issues with the primary antibody and isotype control as well.
Please let me know your results.
I look forward to hear back from you and assist you further in resolving his problem.

Hi,

I recently purchased EGF antibody (ab9695).

I am trying to find the specific referrence in PUBMED of how EGF was used to stain skin with immunohistochemistry. is it possible to sent me that rererence or the papaer so that I can read their staining methods?

Thanks so much in advance for looking into it!

Ioanna

ANSWER:

Thakn you for your enquiry.

I can confirm the following procedure was used to laboratory test this antibody on skin samples to provide the image on the datasheet. Please note this should be a guideline only and may require further optimization in your laboratory:

Antibody concentration: 1.0 μg/ml

Tissue: Normal Human Skin (formalin fixed, paraffin embedded)

Protocol: Rabbit HRP Polymer Kit (AEC chromogen)

Antigen Retrieval: Proteinase K

Incubation Times: 10 min Proteinase K 20 min 1X PBS/ 0.2% Trinton X 100 30 min Fc Receptor Block 10 min Sniper Protein Block Overnight Primary Antibody @ 4°C 10 min Peroxidase Block 20 min Rabbit Probe 20 min Rabbit Polymer HRP 5-10 min Romulin AEC chromogen Results:

This antibody stained normal skin with 4+ apocrine gland epithelial and secretory cells. Everything else is negative. Ideal staining is at 1.0 μg/ml, yet this antibody concentration range was tested from 1.0 μg/ml- 0.25 μg/ml.

I can confirm that any publications that we are currently aware of will be listed on the datasheet. I'm sorry there are none to provide on this occasion for ab9695 EGF antibody. In many cases, regrettably we do not have access to articles mentioning our products, or subscriptions to the journals that publish them.

I hope this will be helpful. Should you have any further questions, please do not hesitate to contact us.

Can you tell me what "neutralisation" means. Does this mean that it prevents the binding effects of the ligand, replaces the effects of the ligand or another biological effect.

Also can you tell me whether this antibody comes conjugated to a flurophore?

ANSWER:

Thank you for your enquiry.

I have been in touch with the source of this antibody and I can tell you that ab9695 has been tested and shown to neutralise the specific activity of the antigen (EGF). The neutralisation is measured by ND50. ND50 is the amount of antibody to neutralize 50% of the biological activity of the specific concentration of antigen.

Unfortunately this antibody is not supplied conjugated to flurophores.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.