Anti-Actin antibody - Loading Control (ab1801)

WB with mouse brain samples
former lot 920447 worked very well (1/1000, 30 min got good band)
this lot GR60919 gives weak band at 1/1000 2 h, 1/2000 no band

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number xxx.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

We have decided to
benefit from our previous codes. We want to have the anti PSD-95 primary
antibody (ab90426) that you have recommended for free. Is there anything
we should do further to have this antibody other than writing to you?

We were wondering if it is possible to benefit from our code for actin
antibody, too. As you may remember from my experiment report that I sent
you yesterday, we could obtain two bands with anti-actin primary antibody,
one band around 55 kDa and one band at 42 kDa. Could you recommend an
alternative antibody for actin and can we have it for free by benefiting
from our code? We may try obtaining one band at 42 kDa this way.
sample was Zebrafish brain lysate.
Thank you very much.

ANSWER:

Thank you very much for your emails, and for your patience.

I apologize for any confusion, but in order to use the codes you can just order the antibodies through your normal method. I believe you have used our distributor, so you can send them your order and include the codes. I have updated the codes so they will be valid for an extra day to allow time to place your order.

Regarding the actin antibody, two band are sometimes seen with ab1801 in certain species, and we believe the antibody is detecting two different forms of actin. The 42 kDa band corresponds to beta actin, the loading control protein. If you would like to try a different antibody as a loading control, we do have ab6046 which has been tested and is guaranteed to work in zebrafish-

http://www.abcam.com/beta-Tubulin-antibody-Loading-Control-ab6046.html

For beta actin, we don't have any antibodies that are guaranteed to work in zebrafish but I would recommend trying ab8226, which was raised against an immunogen that is 86% identical to the zebrafish beta actin sequence-

http://www.abcam.com/beta-Actin-antibody-mAbcam-8226-Loading-Control-ab8226.html

I hope this information will be useful, but please let me know if you have any questions and I will be happy to help you further.

Thank you for your prompt reply. I have submitted our Abreviews for 3
primary antibodies. However I am now aware of not having included testing
codes to our Abreviews.
I have attached more detailed reports of our lab to this e-mail regarding
our western blot experiments. Results with anti Actin and anti
Synaptophysin primary antibodies are fine but results with anti PSD-95
primary antibody are inconclusive for Zebrafish brain lysates. Can you
offer us another anti PSD-95 primary antibody among your companies
products? One more question, is it possible for you to give a code for the
new anti PSD-95 antibody so that we may report whether it reacts with
Zebrafish proteins or not. And if it does not work either can we have a
right to try another antibody for free for third trial?
Thanks in advance,

ANSWER:

Thank you for contacting me and letting me know about this situation.

I have entered your Abreview codes into the corresponding reviews, so these are now active and can be redeemed for free primary antibodies.

I am sorry to see that the results with the PSD-95 antibody were inconclusive. You can use one ofthe testing codes to get a different PSD-95 antibody for freeif you would like, and ab90426 would be my recommendation to try next. In order to provide a code to test an antibody, you will need to purchase the test antibody at full price, so I can not issue a code for ab90426 if you will be using one of the older codes to get ab90426 for free. So, I can either issue a code for ab90426 and you can purchase this at full price, or you can use one of your older codes to acquire ab90426 for free. Please let me know which option you would like, or if you have any questions about this process.

I hope this will be helpful, but please let me know if there is anything else that we can do for you.

Hello,
We have tried troubleshooting suggestions from Abcam about antibodies and
we want to submit Abreviews. Can you please explain how to submit an
Abreview?
Thanks in advance,

ANSWER:

Thank you for your reply.

You can submit an Abreview by logging into your Abcam account and clicking on "Abreviews" on the left side of the screen underneath "My Account". Then, click on "Write an Abreview" and select the options for your review.

You can also submit a review from the datasheet of each antibody. On the datasheet for the antibody you have used, go to the "Abreview" tab and then follow the link to "Submit an Abreview". Then all you need to do is select the application and sample species.

After that you simply need to work through the form, and make sure to include each testing code in the "Additional Notes" section of each review. Once the code is added to the review, it will be active and can be submitted with your order through Kimera for a free antibody per code.

I hope this information will be useful, but please let me know if you have any further questions or need anything else, and I'll be happy to help.

I am attaching extra
document related with my experiment, so that it may be easier to imagine
my result and protocol. Primary antibodies were diluted as suggested. They
were diluted in 5% milk powder (in Tbs-t). My questions are as follows:
1)Experiment with synaptophysin gave intense band results. We are planning
to dilute the primary antibody further. Do you have any suggestion?
2)Experiment with actin gave 2 clear bands. One of them is the expected
band (42 kDa). Do you have a suggestion or a comment?
3)We could not see any hint of a band with PSD-95 experiment. Would you
suggest anything about this issue?
Thabk you in advance,

ANSWER:

Thank you for contacting us and for your patience while I have reviewed the situation. I am sorry to see that the resultshave not been optimal with these antibodies, but I am hopeful that we can improve the blots.

1)With the synaptophysin antibody ab23754, there is quite a bit of background. I would recommend diluting the primary antibody as you mention, trying a different blocking buffer like BSA, and including BSA in the antibody diluents.You could also increase the number of washes from 3to 5. If you're using a PVDF membrane, you can try switching to nitrocellulose as this can give less background.

2) For the actin antibody ab1801, BSA has been shown to be a better blocking agent than milk, so I would recommend trying 3% BSA to block and as an antibody diluent. If the specific band around 42 kDa is stronger than the band around 50 kDa (which seems like that is the case), you can further dilute the antibody and/or increase the blocking step to overnight.

3) It is unfortunately possible that the antibody ab99009does not react with the zebrafish protein, as we have not specifically tested zebrafish lysate. You can try increasing the concentration of the primary antibody (for instance trying 1:250-1:300 if you're currently using 1:1000). Try using a little more protein if needed, up to 30 ug per lane.

After submitting Abreviews for each of these antibodies, you will be able to redeemeach discount code for a free antibody. So for instance, if the PSD95 antibody is still giving no band despite troubleshooting, you can use a code to try a different PSD95 antibody, or any other antibody of your choosing.

I hope these suggestions will be helpful. I will be out of the office for the next week, but feel free to respond to this email and one of my colleages will assist you. Have a great week.