Anti-cIAP2 antibody (ab23423)

Hello
I am indeed unsatisfied with the results.
My goal is to get results and i do not really need a refund. What i need is a good WB.
I am under a lot of pressure since the wbs do not work and i need them done very soon.
I ve already bought crel antibody and i will try it first.
If that antibody does not work,i will have to ask you to send me another one that you think will work.
Thank you

ANSWER:

Thank you for your reply, and we are sorry that the results have not been satisfactory.
My colleague is out of the office today, however she will get back to you as soon as she returns. Please keep us updated about the results with the other c-Relantibody.

Please let me know if you have any questions and I'll be happy to help.

Actually I tried both incubating with secondaries separately and jointly.

There was no difference so far.

I don't know how those antibodies would compete with each other since they are against different hosts. One is anti rabbit another one is anti mouse.

Ive been doing ICC and IHC for quite a time and never had that problem.

ANSWER:

Thank you for your reply.

Many antibodies will be suitable for simultaneous incubation, however, some antibodies will interfere with each other's binding. For example, if an antibody hasa weaker binding affinity, it is often observed that incubation in a solution that contains a high concentration of other proteins can prevent effective binding. For this reason, it is often recommended that blocking agents in the antibody diluent be kept at a concentration of 1% or lower. Incubating multiple primary antibodies simultaneously may result in reduced binding efficacy.

As I mentioned earlier, if you are unsatisfied with the results, please let me know and I will be happy to offer you a refund or credit on your original order.

Have you gotten a chance to take a look at the attached pictures?

1. I load maximum. 40-50micrograms
2. I always use RIPA buffer
3. 5% BSA was used in both cases.
4. Primary overnight , I also tried over the weekend. Secondary 1-2 hours
5. secondaries were from GE dluorescent anti rabbit and anti mouse cy5 and cy3 respectively. Yes they work perfectly as u can see in the ppt. although only cy3 is shown there as positive control.
cy5 worked with my other antibodies.
6. Images were exposed at list to 5-10 minutes. as long as it allows me. I didnt see anything.
7. I m not really sure what was the original order number since I didnt order the antibodies myself.

ANSWER:

Thank you for your reply and for clarifying your protocol details.

When you say that you saw a weak band the first time you ran the gel, are you stripping and reprobing the same membrane? There will be some protein loss each time the membrane is stripped and reprobed and that might account for the weak signal.

Is the c-Rel antibody in the image ab108299 or the replacement I had sent you, ab83094?

When you are performing the immunostaining, are the two antibodies incubated simultaneously or serially? If the antibodies are incubated simultaneously, it is possible that they may interfere with each other's binding. This is often observed in double staining in ICC. Have you tried these antibodies on their own?

I hope this helps. If you are unable to improve your results, please let me know and I will be happy to offer you a refund or credit of your original purchase.

Recently I contacted abcam concerning antibodies that I purchased.

Last Birc3 antibody worked once at 1 in 250 concentration. I had relatively strong band after long exposure.

Although it didn't work next time. I could not even get the band in the positive control.
Instead as you can see in the picture in the ppt slide attached there is a very strong 50kDa band (i used even higher concentration 1 in 100 to make sure I get a strong band).

cREL showed a weak band in the Hela lysates at 1 in 100 concentration even though the method that I use is more sensitive than chemiluminescence.

I used all 50 microliters in the first time. I kept the diluted antibody and re used it. I tried the antibody with oxidative stressed nuclear extract samples that I had in order to see whether I can detect a stronger band.

Unfortunately there was no band detected even in the HELA lysates. As you can see the TBP is there. TBP is from abcam as well and it works great.

I cant get any data and unfortunately I need that data very soon.

I hope we can solve this problem.

ANSWER:

Thank you for contacting us. I am sorry that these antibodies are not giving satisfactory results in your western blot. Could you please provide some additional details about your protocol?

1)Howmuch protein was loaded in the gel?We typically recommend loading 20-40ug of protein per lane to ensure agood signal.

2)What lysis buffer was usedto prepare your samples? Becausec-Rel is a nuclear protein, a lysis buffer containing both ionic and non-ionic detergents (such as RIPA buffer)will ensure that the c-Rel in your samples is released effectively.

3)What blocking agent was used in your experiment?Some antibodies maybe sensitive tospecific blocking agents.Our cIAP2 antibodyab23423was validated using 3% BSAas a blocking agent, while the c-REl antibodyab108299 was validated using 5% milk.

4) What were the primary and secondary antibody incubation times?It can often help to increase signalto incubate overnight at 4C with the primary antibody.

5) What secondary antibodies were used and at what dilutions? Have these secondaries beenworking well with other primary antibodies?

6) How long were theimages exposed? Wereyou able to see any bands at the correct molecular weights by overexposingthe blot?

7) What was your original orderor PO number?

I hope thiswill help to improve your results, ifnot, please let me knowand Iwill be happy to assist you further.

Our lab has recently purchased the cIAP2 antibody (ab23423) for westerns (our westerns are on Peripheral Blood Mononucleocites extracted from Blood and Spleen). I was wondering if you have a positive control for sale or have any suggestions for a good positive control that we can use?　Thanks,

ANSWER:

Thank you for your enquiry.

For western blot, HeLa cell lysates can be used as a positive control, as seen in our laboratory testings (datasheet WB image).

Also, according to SwissProt (http://www.uniprot.org/uniprot/Q13489), the protein is highly expressed in fetal lung, and kidney. In the adult, expression is mainly seen in lymphoid tissues, including spleen, thymus and peripheral blood lymphocytes. Therefore, these could also be used as a positive control.

I hope this information will be useful. If there is anything that I can help you with, please do not hesitate to contact me.

Have a nice day!