Anti-NG2 antibody [LHM 2] (ab20156)

Good afternoon,

I bought 2 antobodies from Abcam:

- Anti-NG2 (LHM 2) - ab20156
- Anti-PDGF receptor beta - ab32570

I need some informations.



Can you tell me if I need to permeabilize the cells?

Are these membrane antibodies?



Thank you

Patricia
--

Patricia Pitrez Pereira, PhD Student



MIT Portugal Bioengineering Systems
http://www.mitportugal.org/


Biocant - Center of Innovation and Biotechnology

Nucleo 4, Lote 3
3060-197 Cantanhede - Portugal

http://www.biocant.pt/
Phone: +351 231 419 040
Fax: +351 231 419 049
http://biomaterials.biocant.pt/

ANSWER:

Thank you for your email and for your telephone enquiry earlier today.

As discussed on the telephone, PDGF receptor beta is a membrane protein, and so I would recommend that detection with ab35270 would not usuallyrequire permeablization. Though this may require some individual optimization.

NG2 is an apical cell membrane protein. Reviewing the details after our phone conversation,as this is a membrane protein, it will not require permeabilization. However, as this protein can alsorelocate to other parts of the cell, such as thelamellipodia, I can recommend you may require a gentle permeabilization step. A strong detergent like Triton may not be suitable, as this can disrupt membrane proteins and significantly affect the staining. I can suggest it would be more appropriate to include more gentle detergent such as 0.2% Tween or saponin in the antibody dilution buffers and wash buffers. This will help to keep the cells permeabilised. Again, this is a guideline only and may require some individual optimization.

Usually for membrane proteinswe would not recommend to use organic solventsto fix the samples, as this also permeabilizes the cells and can cause disruption to antibody epitopes on membrane proteins.However, I do notice that an Abreview on teh datasheet forthe NG2 antibody indicates that acetone fixation worked best in frozen sections. This does highlight and indicate that although we canprovide recommendations, individual optimization is sometimes required.

http://www.abcam.com/index.html?datasheet=20156&tab=abreviews&intabreviewid=6408

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

May I test these in rabbit?

ANSWER:

DISCOUNT CODE for AB85308: Expiration date:

I am very pleased to hear you would like to accept our offer and test ab20156 and/or ab85308 in IHC on rabbit. This code will give you 1 free PRIMARY ANTIBODY before the expiration date. To redeem this offer, please submit an Abreview for IHC in rabbit and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

The spec sheet says it works on paraffin sections. What tissue was used to show that this antibody works? What concentration of antibody? Does the antibody require antigen retrieval of the tissue?

Thanks

ANSWER:

The antibody was tested in IHC on keratinocytes in the epidermis in the paper Ghali L et al. Epidermal and hair follicle progenitor cells express melanoma-associated chondroitin sulfate proteoglycan core protein. J Invest Dermatol 122:433-42 (2004). PubMed: 15009727

I'm afraid I do not have access to this paper to find out the protocol used to test the antibody. I suggest you try a range of dilutions and incubation times (1:50,1:100, 1:200, 1:500, 1:1000 for example).

The following references have also used this clone: Kupsch JM et al. Isolation of human tumor-specific antibodies by selection of an antibody phage library on melanoma cells. Clin Cancer Res 5:925-31 (1999). PubMed: 10213230

Kupsch JM et al. Generation and selection of monoclonal antibodies, single-chain Fv and antibody fusion phage specific for human melanoma-associated antigens. Melanoma Res 5:403-11 (1995). PubMed: 8589614

I hope these references will help,