Anti-GFAP antibody [2A5] (ab4648)

Thank you for contacting Abcam.

Earlier we discussed your sELISA with ab4648 as the capture and ab48050 as the detector. As we discussed, since you would like to use your streptavidin-gold conjugated secondary, a detector that is conjugated to biotin is preferable so you can obtain the signal amplification necessary to have a detectable signal. Thus, we discussed testing ab79203 as your detector antibody through our AbTrial program. Your discount code and information about the abtrial program is given below:

I am very pleased to hear you would like to accept our offer and test ab79203 in sELISA. This code will give you $XXX off your next order before the expiration date. To redeem this offer, please submit an Abreview for sELISA and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active. 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: https://www.abcam.com/abtrial

On the phone I forgot to mention our current RabMab promotion, which entitles you to a free rabbit monoclonal antibody with any purchase of a primary antibody. Quote “RABMAB-XBSMG” in your next primary antibody order to take advantage of this offer. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

The protocol used was as follows:
Buffers and Reagents:
Carbonate Coating Buffer (100 mM)
Antigen or antibody should be diluted in coating buffer to immobilize them to the wells:
3.03 g Na2CO3,
6.0 g NaHCO3
Make up to 1000 ml with distilled water and adjust to pH 9.6
PBS:
1.16 g Na2HPO4,
0.1 g KCl,
0.1 g K3PO4
4.0 g NaCl
Make up to 500 ml with distilled water and adjust to pH 7.4
Wash Solution - PBST (0.1% (v/v) Tween):
Add 5ml 10% (v/v) Tween-20 stock solution to 500ml PBS,
Blocking and Antibody/Protein Dilution Buffer:
5% (w/v) BSA in PBST.
Stop Solution:
0.25M Sulphuric Acid
Protocol:
1. 96-well ELISA plates are coated with 100µl/well of Capture Antibody ab4648 diluted 1:5000 in 100mM Carbonate Coating buffer, pH9.6.
2. Incubate the plates overnight at 4°C or 1 hour at room temperature to allow adsorption of the Capture Antibody to the plate.
3. Remove the Capture Antibody and was the plate 3 times with PBST (3x300µl/well), leaving the plate dry after the final wash.
4. Block the plates with 150µl/well of Blocking Buffer.
5. Incubate plates at room temperature for 1 hour.
6. Aspirate Blocking Buffer and add 50µl/well of Target Protein at the recommended concentration in Blocking Buffer.
7. Incubate for 1 hour at room temperature.
8. Remove the Protein Solution and was the plate 3 times with PBST (300µl/well), leaving the plate dry after the final wash.
9. Add 50µl/well of Detector Antibody ab48050 diluted to 0.5 ug/mL in Blocking Buffer.
10. Incubate for 1 hour at room temperature.
11. Remove the Detector Antibody solution and wash the plate 3 times with PBST (300µl/well), leaving the plate dry after the final wash.
12. Aliquot 100µl/well of anti-rabbit HRP conjugate Secondary Antibody diluted 1:10,000 in Blocking Buffer.
13. Incubate for 1 hour at room temperature.
14. Remove the Detector Antibody solution and was the plate 3 times with PBST (300µl/well), followed by 2 washes with PBS, leaving the plate dry after the final wash.
15. Add 50µl/well of TMB Peroxidase Substrate.
16. Incubate for 10 minutes at room temperature (The length of this incubation may be optimised, but 10 minutes works well in most cases).
17. Add 50µl/well of 0.25M Sulphuric Acid Stop Solution.
18. Read plates on plate reader at 450nm.

We do recommend usingthe Sodium Citrate buffer (pH 6.0), however HIER are effective as well.