Question (16316) | Anti-CSN2 antibody (ab10426)

Go to datasheet (ab10426)

Question

We ordered two samples of anti-CSN2 (ab10426) last May. Our Purchase Order number was CAMPANERO-0161. It seems there was some stock shortage and we received the second sample 1-2 months after the first one. This is likely the reason because these two samples were from different lots (lots numbers 114133 and 82446). We obviously started by using the first lot that we received (114133) and noticed that it worked out well in immunoblotting experiments designed to detect the endogenous protein in samples derived from human cell lines as well as in experiments designed to detect ectopically expressed CSN2. As this lot was getting finished, we started to use the second lot and found no specific signals. At the beginning we thought we were doing something wrong, but now we designed an experiment with the only purpose to compare both lots and our results clearly indicate that this second lot (82446) is not working at all. We have carried out two different experiments to compare both lots performance. In the first experiment (see enclosed file with Fig 1a and 1b), we immunoblotted extracts from cells transfected with either a mock vector or with a vector encoding a GST fusion with either CSN2 alpha or CSN2 beta. This nitrocellulose membrane was probed first with anti-CSN2 (lot 114133) and second with anti-GST. As you can see, the anti-CSN2 antibody (lot 114133) reacts with both the GST fusion proteins and the endogenous CSN2 alpha (Alien alpha) while the anti-GST Ab only recognizes the GST fusion proteins. In the second experiment we transfected Saos-2 cells with different expression vectors for CSN2 (Alien) alpha or beta tagged with either HA or GST. Please, note that pMT2-driven Alien alpha is expected to be larger than the pcDNA3-driven version. Cell extracts were immunoblotted and the membrane was sequentially probed with anti-CSN2 (lot 82446), anti-HA, and anti-GST. As you can see in Fig. 2 (see enclosed file), the anti-alien Ab was unable to react with any of the different transfected CSN2 forms at all, while the anti-HA recognized the HA-tagged versions of Alien alpha and beta and the anti-GST Ab reacted with the GST-tagged CSN2 alpha and beta isoforms. Please, notice that we used the same extracts for lanes corresponding to GST-Alien alpha or GST-Alien beta that we used in Fig 1. We believe these results clearly demonstrate that anti-CSN2 ab10426 (lot 82446) fails to work as expected. Unfortunately, we have lost a lot of time and wasted a lot of reagents by using this antibody and we would like, at least, to receive a sample that works fine from a different lot. Finally, according to antibody ab10426 datasheet, this antibody is predicted to react with Mouse due to sequence homology. In this regard, we wanted to let you know that, at least in our hands, this antibody fails to recognize the mouse CSN2 protein and we wonder if you have any new information about tests performed with this or with any other anti-CSN2 antibody on mouse proteins.

Answer

Thank you for your enquiry. May I also thank you for explaining the problem so well. The images that you e-mailed are beautiful and clearly highlight the problems that you have been having. We often observe LOT variation in our antibodies. This can be attributable to the preparation of the antibody. From the results that you have shown me I can clearly see that there is an element of LOT variation. Whilst you are detecting both the endogenous CSN2 (Alien) and ectopically expressed GST tagged forms of the protein with LOT number 114133 you are only detecting the endogenous forms of the protein with LOT 82446. Quality is important to Abcam and I thank you once again for highlighting this to us. Unfortunately I cannot provide you with a replacement vial as your purchase falls outside of our 90 day refund and credit note policy. We currently have two lots in stock 114133 and 114751. Were you wishing to place an order you are welcome to specifically request LOT 114133 given that this has behaved well in your experiments. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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