Anti-Nestin antibody [196908] (ab6320)

Thank you for your reply with this information.


Regarding your protocol - one thing that really stood out is the use of Triton as a permeabilizing agent. This detergent will also partially dissolve the nuclear membrane and will disrupt proteins when used at higher concentrations for longer periods of time. We typically recommend using it at 0.1-0.2% for 10 min only. Since both of these proteins are cytomplasmic, permeabilization with tween 20 is sufficient (I recommend 0.2 % for 10 - 15 minutes). This should decrease the amount of background and non-specific stain you are observing.


Additionally, I would recommend optimizing the conditions with each antibody individually rather than in sequential staining.


I hope this information is helpful. Please do not hesitate to contact me if you have any additional questions or concerns.

Thank you for your enquiry. Ab27952 is a rabbit polyclonal antibody to nestin, and ab6320 is a mouse monoclonal antibody. The immunogen used to generate ab27952 was a KLH conjugated peptide to the human nestin tail. The immunogen used to generate ab6320 were nestin-transfected NS0 cells transfected with a human Nestin fragment aa residues 618-1618. Ab27952 detects human and mouse nestin while ab6320 detects human nestin and has no reactivity with rodent nestin. Please contact us again if you have any additional questions.

Thank you for your enquiry and your interest in our products. There is no specific protocol for this anti-Nestin monoclonal antibody. Whatever cell line is being studied, the cells must be permabilized and fixed for FACS. Here we send our general suggested protocol for Flow Cytometry: Materials and Equipment Sample cells Vortex mixer 12x75mm test tubes Flow cytometer PBS/0.1% sodium azide/ 1% FBS PBS, 0.5% Paraformaldehyde, 4° C Fluorescent-labeled Antibody Pipettes Centrifuge Ice PBS/ sodium azide at 4o C Preparation of Cells 1. Prepare the desired biological cells according to the appropriate protocol. Adjust the concentration of the cells to 2x10e7 cells per ml (or 1x10e6 cells per 50ul: each test consists of 1x10e6 cells), diluting with PBS/0.1% sodium azide / 1% FBS or BSA, as necessary. The cells can be isolated up to 4 hours before being stained as long as they are kept on ice. Note: In some cases, use of a non-specific binding or blocking agent may be desired. If so, add the blocking agent and incubate at RT for 10 minutes prior to the addition of a antibody to the cells 2. Dilute the fluorophore conjugated antibody appropriately according to the specific product recommendation for the staining being done. Use PBS/0.1% sodium azide/ 1% FBS for the dilution. Typically, 0.125-0.5ul of conjugated monoclonal antibody stains 10e6 cells. Dilute the antibody so that a volume of 10-20ul are added to the cells. 3. Pipette 50ul of the previously isolated biological cells into the bottom of each labeled 12x75mm test tube. Add the appropriate volume of diluted antibody and mix gently. Incubate the cells with the antibody at 2-8° C for 30 minutes in the dark. All conjugated antibodies ( FITC, R-PE, etc.) for double or triple staining can be added simultaneously at this point and do not require additional incubations. 4. After incubation remove the unbound antibody from the cells by washing with 1ml of PBS/0.1% sodium azide/ 1% FBS. Pipette the PBS/0.1% sodium azide/ 1% FBS into each tube, vortex, then centrifuge at 350xg for 5-7 minutes at 2-8°C. Carefully aspirate the supernatant leaving about 50-100ul in the bottom of each tube. Repeat this process for a total of 3 washes. 5. If flow cytometry is to be performed the same day, resuspend the cells in 0.5ml of cold PBS/0.1% sodium azide/ 1% FBS after aspirating the supernatant. Gently vortex and analyze the cells. If analysis will not be performed the same day, the cells may be fixed in 0.5ml cold PBS with 0.5% paraformaldehyde and store at 2-8 °C in the dark in buffer containing 0.1% sodium azide for as long as 48 hours. Indirect Staining of Extracellular Proteins If an unconjugated antibody is being used with double or triple staining then the primary antibody should be incubated for 30 minutes at 4 °C and removed by washing with PBS/0.1% sodium azide/ 1% FBS. At the time, add any conjugated antibodies and repeat staining protocol. In general, direct staining is overwhelmingly preferable. Flow Cytometry for Intracellular Antigens Similarly, a collection of cell types can be sorted of the basis of specific intracellular antigen using Flow Cytometric methods. Cell Fixation and Permeabilization 1. Wash the cells twice with cold PBS. Temperature control at this point is of vital importance. Note: Cells should be kept on ice anytime they are not spinning and centrifugation in a thermostated centrifuge at 4 °C is greatly preferred. 2. Fix the cells with 1% paraformaldehyde in cold PBS for 15 minutes (4 °C). It is very important to assure that the cells are uniformly resuspended during fixation. 3. Wash the cells twice with cold PBS. 4. Permeabilize the cells with 100% ice-cold methanol added dropwise while the cells are gently vortexing. Again, it is vitally important that the cells are uniformly suspended. Allow the soak to soak in cold methanol for 15 minutes. 5. Wash the cells twice with cold PBS. Antibody Incubation 1. Add approximately 1 x 106 cells to each flow cytometry tube and wash with 1 ml of 0.1% saponin or Tween 20 diluted in PBS with 2% FBS added. 2. Resuspend cells in 1 ml 0.1% saponin(Tween 20) + 2% FBSand allow to incubate for 30 minutes blocking at room temperature. This step is to block non-specific binding. 3. Add 20 ul of antibody diluted to the recommended concentration in 100 ul of the above blocking solution and incubate for 20 minutes at room temperature. 4. If the primary antibody is not conjugated with a fluorochrome, then a second incubation with a fluorochrome-conjugated secondary antibody will be necessary. Incubate 20 minutes in blocking solution at room temperature. 5. Wash the cells once with blocking buffer, then finally with PBS. 6. Resuspend the cells in 500 ul PBS and run on flow cytometer. We hope this information will be useful for you.