Anti-Rb antibody (ab6075)

Dear Kate

Here's the customer reply to your questions, please advise:


With the TBP antibody ( ab23474 ), we are using L6 cells and these are of rat origin. Would this be the reason for the negative results? I do not have enough antibody left to check any further tests and currently I do not work on human cytoplasmic and nuclear extracts. I need some marker for nuclear extracts that can be used on protein lysates obtained from rat/mouse origin.

Please advice.

Thanks again for your kind help.

Regards,

ANSWER:

Thank you for your reply and for kindly providing these details.

Regrettably, I am sorry ab23474 Anti-TATA binding protein TBP antibodyis not covered by our guarantee for rat samples. I appreciate your concerns and it is regrettable the results have not been successful on this occasion.

If you wish to use a nuclear marker in rat and WB, then the following Rb protein antibodies should be more suitable and would be covered by our 6 month guarantee for this purpose. I can recommend toreview the online datasheets for further information:

ab6075 Anti-Rb antibody
Rabbit polyclonal
ICC/IF, IHC-Fr, IHC-P, IP, WB
Reacts with: Human, Mouse, Rat
http://www.abcam.com/index.html?datasheet=6075 (or use the following: http://www.abcam.com/index.html?datasheet=6075).

ab85607 Anti-Rb antibody
Rabbit polyclonal
ICC/IF, IHC-P, WB
Reacts with: Human, Mouse, Rat
http://www.abcam.com/index.html?datasheet=85607 (or use the following: http://www.abcam.com/index.html?datasheet=85607).

I hope this will be helpful to you.

je voudrais utiliser les anticorps dont les références sont :
Anti-Rb antibody (ab6075),
Anti-Cdk2 antibody (ab7954),
Anti-Cdk4 antibody [DCS-35] (ab3112).
Je ne sais pas si je peux avoir un échantillon afin de pouvoir les tester puis les commander s'ils me conviennent ou si cela n'est pas possible, avoir une offre découverte.
Je vous remercie d'avance,

ANSWER:

Notre catalogue contient plus de 70 000 références anticorpset nous ne pouvons malheureusement pas offrir, pour des raisons logistiques et de stockage, d'échantillon gratuit ou de petit volume pour des essais préliminaires.

Nos produits sont garantis pour fonctionner tels qu'ils sont décrits sur les fiches techniques (garantie Abpromise de 6 mois). Si vous rencontrez des difficultés avec un de nos produits nous pourrons résoudre le problème en vous apportant une aide technique,en vous remboursant, ou en envoyant un anticorps de remplacement (autre lot ou produit alternatif).

Si vous désirez utiliser un anticorps contre une espèce ou dans une application non testées préalablement, nous pouvons vous offrir une offre de test. Il suffit de contacter notre service scientifique par email mailto:supportscientifique@abcam.com .

J'espère que ces informations vous seront utiles. N'hésitez pas à nous contacter de nouveau si vous avez d'autres questions.

The customer called to inquire as to whether our anti-RB products would also recognize RB2 and RB3 in rat on western blot.

ANSWER:

Thank you for contacting Abcam and for your help in finding the associated proteins on the phone.

I have had the chance to run the alignments of these proteins and have found that the homology between them is extremely poor. As such I do not believe that you will be able to find a single product which recognizes all targets. I have compiled a short list of products which re against each protein and are suitable for western blot on rat tissues for you.

RB1 (alternative names include:p105-Rb,Osteosarcoma)

ab6075:http://www.abcam.com/Rb-antibody-ab6075.html

ab85607:http://www.abcam.com/Rb-antibody-ab85607.html

RB2 (alternative names include: Rb2 p130, RBL2)

ab6545:http://www.abcam.com/Rb2-p130-antibody-ab6545.html

ab17124:http://www.abcam.com/Rb2-p130-antibody-130P215-ab17124.html

RB3 (alternative names include:Stathmin like 4, STMN4)

ab110521:http://www.abcam.com/STMN4-antibody-ab110521.html



I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

1) Abcam product code ab6075




2) Abcam order reference number or product batch number



3) Description of the problem

Detection of 3 bands smaller than 100kDa



4) Sample preparation:

Type of sample (whole cell lysates, fraction, recombinant protein…): Adrenal tissue and cell lysate

Lysis buffer RIPA

Protease inhibitors:

Phosphatase inhibitors Aprotinin, Leupeptin and PMSF

Reducing agent

Boiling for ≥5 min? yes

Protein loaded ug/lane or cells/lane 30ug/lane

Positive control MCF7 cells lysate

Negative control



5) Percentage of gel 8%

Type of membrane Nitrocellulose

Protein transfer verified Yes, Ponceu

Blocking agent and concentration Milk 5%

Blocking time 2 hours

Blocking temperature RT



6) Primary antibody (If more than one was used, describe in “additional notes”) :

Concentration or dilution 1:1000

Diluent buffer TBS+1%tween+5%milk

Incubation time 12 hours

Incubation temperature: 4°C



7) Secondary antibody: Amershan ECL

Species: Rabbit

Reacts against: Rabbit

Concentration or dilution 1:1000

Diluent buffer TBS

Incubation time 1 Hour

Incubation temperature: RT

Fluorochrome or enzyme conjugate: Horse-radish ECL



8) Washing after primary and secondary antibodies:

Buffer TBS+1% Tween

Number of washes 3



9)Detection method

10) How many times have you run this staining? Two

Do you obtain the same results every time? No

What steps have you altered to try and optimize the use of this antibody? We increased the block time



Document attachment: Attaching images of your blot is strongly recommended and can greatly speed up our investigation of your problem.

ANSWER:

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.


Reviewing this case, I would like to offer some suggestions to help optimise the results from ab6075.

1.) The band that can be seen in the positive control (MCF7)is at the right height in my opinion. This means we just have to deal with additional bands.

2.) According to the following publication there are splice variant of this protein that correspond insize to the bands seen in adrenal samples:

Q. Ping Dou and Bing An, Rb and apoptotic cell death. Frontiers in Bioscience 3, d419-430, April 6, 1998. http://www.bioscience.org/1998/v3/d/dou/d419-430.htm

3.) I suggest to prepare nuclear fractions to clean up the blot and find out which bands corresponds to the nuclear Rb protein.

4.) To ascertain if some of the additional bands are due to the secondary antibody cross- reacting, I suggest to run a "no primary" control.

5.) As an additional positive control, I recommend to use the following cell lines, which we have use to test this antibody: A431 and K562

I would also appreciate if you can confirm some further details:

Could you please confirm how the antibody was stored?

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

Hi there, I was wondering if you could let me know if you have any pure positive controls or blocking peptides for either of these two antibodies:

Anti-Phospho Rb (T821) #ab4787 Anti-Rb (ab6075)

For both there are suggested cell lysates to use as positive controls, but I need a pure protein to verify the exact band on my western blots. I know you provide a His-tagged version of the Rb full length protein, but do you have any non-tagged versions? Or do you know a good vendor for these? Also, the blocking peptide for phospho-Rb is no longer available (#ab5324); is there another source I could use to get this blocking peptide?

ANSWER:

Thank you for contacting us. We currently only have the tagged Rb proteins in our catalog and no blocking peptide for ab4787 is available. I would sugges Biocompare.com to search for these products.

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