Anti-RbAp48 antibody [11G10] (ab488)

We have used the AB 488 antibody to immunoppt RbAp48. In cell lysates a rabbit or goat anti-RbAp48 antibody detects one band but after IP, two bands are observed, with a second slower migrating band quite close to RbAp48 appearing. It appears that others have had similar experiences with this antibody. Do you know what is the slower migrating band?

ANSWER:

Thank you for your inquiry.

We have heard similar comments from customers.

What species are your cell lysates from? And have you pre-cleared your lysates?This would bepre-incubating the prepared lysate with the beads before commencing with the immunoprecipitation (please see the protocol linked below). This should clear the lysate of any proteins that are binding non-specifically to the beads. Some researchers also use an irrelevant antibody of the same species of origin and same Ig subclass to pre-clear the lysate.

http://www.abcam.com/index.html?pageconfig=resource&rid=11385

There are 4 isoforms of RBAP48 according to SwissProt (http://www.uniprot.org/uniprot/Q09028). The extra band may be anisoform. Or on top of that it could be a phosphorylated form of the protein.

Also, the RBAP48 protein is 87% similar with RBAP46, so it may be cross-reacting. We unfortunately haven't done any further test with this antibody so I am sorry we will not be able to confirm it.

Or, it could be theheavy chain that theanti mouse secondaryisdetecting(50-55kDa). We would strongly recommend running an isotype control.

I hope this information helps. Please contact us with any other questions.

I have
some questions regardiing the product RbAp48 antibody(ab488).
I am doing an Immunoprecipitation of RbAp48 with this antibody from
HeLa cell lysate. The IP worked very well (as you can find in the
attached file), but in the IP fraction, this antibody has detected two
bands (lane 2). I have used the mouse IgG as a control. In the control
sample, it has also detected two bands, I guess this is corresponds to
mouse IgG, as the sizes are very similar with RbAp48, it is hard to
distinguish. How ever, whenever I use the rabbit RbAp48 antibody, it
is not detected in control sample. Please let me know if I am right or
not.
My most important concern is in the IP fraction this antibody is
detecting two bands as I mentioned earlier (does not matter which
antibody I use, mouse or rabbit). I want to know if you have any
information regarding this.
Could the upper band be RbAp46? ( if this antibody can detect both
RbAp48 or RbAp46?). Another possibility if the upper band could be
phosphorylated RbAp48 and whenever we enrich RbAp48 by doing IP, It
can detect the hyperphosphorylated RbAp48 but not in the input
sample? These are some concerns for which I need to be confirm.
As We are preparing a manuscript for publication, this needs to be
clarified. Please let me know as early as possible.
Thank you so much in advance for your help and cooperation.
Regards

ANSWER:

We have previously been in contact regarding ab488, which was not providing satisfactory results.

We provided some troubleshooting tips for the protocol, and I am writing to inquire if these suggestions have helped to improve the results? I would be pleased to receive an update on how these experiments are progressing.

If the antibody is still not working as stated on the datasheet, and the item was purchased within 6 months of contacting us, we would be happy to replace or refund the antibody.

I wish you the best of luck with your research and I look forward to hearing from you. Please do not hesitate to contact us if you have any further questions.

Hi there, I have some questions regardiing the product RbAp48 antibody(ab488).

I am doing an Immunoprecipitation of RbAp48 with this antibody from

HeLa cell lysate. The IP worked very well (as you can find in the

attached file), but in the IP fraction, this antibody has detected two

bands (lane 2). I have used the mouse IgG as a control. In the control

sample, it has also detected two bands, I guess this is corresponds to

mouse IgG, as the sizes are very similar with RbAp48, it is hard to

distinguish. How ever, whenever I use the rabbit RbAp48 antibody, it

is not detected in control sample. Please let me know if I am right or

not.

My most important concern is in the IP fraction this antibody is

detecting two bands as I mentioned earlier (does not matter which

antibody I use, mouse or rabbit). I want to know if you have any

information regarding this.

Could the upper band be RbAp46? ( if this antibody can detect both

RbAp48 or RbAp46?). Another possibility if the upper band could be

phosphorylated RbAp48 and whenever we enrich RbAp48 by doing IP, It

can detect the hyperphosphorylated RbAp48 but not in the input

sample? These are some concerns for which I need to be confirm.

As We are preparing a manuscript for publication, this needs to be

clarified. Please let me know as early as possible.

Thank you so much in advance for your help and cooperation.

Regards

ANSWER:

Thank you for contacting us.

There are 4 isoforms of RBAP48 reported in swissprot (http://www.uniprot.org/uniprot/Q09028). The extra band may be isoform of the RBAP48. I would suggest searching literature. Further tests may be needed.

The RBAP48 protein is 87% similar with ABAP46 protein so it is more likely that the lower band is RBAP46. We unfortunately haven't done any further test with this antibody so I am sorry we will not be able to confirm it. To confirm the identity you may need to try an RBAP46 antibody which does not cross react with RBAP48.  

The most possible cause of bands in IgG (which I think is an isotype control) is the heavy chains of beads conjugated antibody.  The anti mouse secondary will detect the heavy (50-55kDa) and light chain (25 kDa) of the mouse primary antibody. The disappearance of bands with rabbit antibody indicates the presence of heavy chains.

Ab488 is not a phospho specific antibody; it will detect both the phospho as well as non phospho form.

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

I am writing to inquire whether both, or either, of these antibodies (ab86 and ab488, mouse monoclonal antibodies against RbAp48) cross react with RbAp46 protein? I am looking for an antibody that will be specific for RbAp48 only.

Also, does the the rabbit polyclonal antibody against RbAp46, ab3535, cross react with RbAp48 protein?

Thank You

ANSWER:

Thank you for your patience. The originator of ab3535 has said that the antibody should not cross react with the RbAP48 protein.

Please contact us again if you have any additional questions.

I am writing to inquire whether both, or either, of these antibodies (ab86 and ab488, mouse monoclonal antibodies against RbAp48) cross react with RbAp46 protein? I am looking for an antibody that will be specific for RbAp48 only.

Also, does the the rabbit polyclonal antibody against RbAp46, ab3535, cross react with RbAp48 protein?

Thank You

ANSWER:

Thank you for your enquiry. For ab86 and ab488, the antibodies have not been tested against RbAp46 so we do not know whether or not the antibodies would react with it. I'm still waiting for information on ab3535 and will let you know when I receive.