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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?
Patricia Pitrez Pereira, PhD Student
MIT Portugal Bioengineering Systems
Biocant - Center of Innovation and Biotechnology
Nucleo 4, Lote 3
3060-197 Cantanhede - Portugal
Phone: +351 231 419 040
Fax: +351 231 419 049
Asked on May 03 2012
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.
I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.
Answered on May 03 2012