Anti-Cyclin D1 antibody [CD1.1] (ab6152)

Thanks for your kindly reply, after contacting with this customer she would like to have a credit note.
Could you please help her to create a credit note?

Thanks for your kindly assistance.

Best regards

ANSWER:

Thank you for confirming this information and for your help and cooperation with this case.

As requested, I have asked our accounting department to issue you with a credit note. This can then be redeemed against the invoice of a future order.

Credit ID: ####

As usual if you have any further questions regarding this credit note, please contact the accounts department by email at creditcontrol@abcam.com. Please refer to the credit ID number in any correspondence with the accounting department.

I would like to wish the customer good luck with their research. The technical team is always at your service, should you require further expert advice.

Thanks for your kindly reply, this customer reply you question as follow:

1. Yes, clear!

2. We used RIPA as a lysis buffer.

5. This secondary antibody can work with other primary antibody well! From cell signaling.

We already tried so many times and also modified our experiment step; we have a lot of experiments to finish, could you please solve this problem as soon as possible?

This customer has a little upset about this state; she wants to have a credit note, if you can issue a credit note to her I will be very appreciate about that.

Thanks for your kindly help

Best regards

ANSWER:

Thank you for your message.

I am sorry to hear the suggestions have not improved the results on this occasion. I fully understand the concerns of the customerand it is disappointing the results have not been successful.

We are happy to offer a refund, credit note or free of charge replacement when a product is not working in a successfully tested applications or species within the guarantee period. However, we do often find that suggesting some scientifically thought out optimization tips helps to improve the results. So I hope you can understand that we like to offer the best technical support possible to provide a satisfactory outcome.

On this occasion, as this antibody is not working as it should I would be pleased to offer an immediate free of charge replacement or credit note.

Thank you for your understanding and your continued cooperation. I look forward to hearing from you with details of how you would like to proceed.

Dear technical support team:

This customer has purchased ab6152 (Anti-Cyclin D1 antibody [CD1.1]) and has conducted the wb several times with human sample. The results show no band, therefore, she would like to ask for your help to modify her experiment step, could you please offer any suggestion to improve her result?

Her experiment step as follow:

1. Order details:


Batch number: gr46418-2

Abcam product code: ab6152

Antibody storage conditions (temperature/reconstitution etc) -20℃

2. Please describe the problem (high background, wrong band size, more bands, no band etc).

We also try the different antibody concentration (1:200, 1:500, 1:1000) on the positive cells and bladder cancer tissue. There are no bands on positive cells (positive cells are provided by abcam sales) and bladder cancer tissue (12 paired samples from 6 people with bladder cancer).we has tested this antibody for different staff. There is still no any band

3. On what material are you testing the antibody in WB?

Species: human and cell
What cell line or tissue: bladder cancer tissue
Cell extract or Nuclear extract: Cell extract
Purified protein or Recombinant protein: Purified protein

3. The lysate

How much protein was loaded: 50ug

What lysis buffer was used:

What protease inhibitors were used: protease inhibitor cocktail tablets(Roche)

What loading buffer was used: 5x sample buffer

Phosphatase inhibitors

Did you heat the samples: temperature and time: 37 ℃ 15min

4. Electrophoresis/Gel conditions/ Transfer conditions

Reducing or non reducing gel:

Reducing agent:

Gel percentage : 12％

Transfer conditions: (Type of membrane, Protein transfer verified): PVDF

5. Blocking conditions

Buffer:

Blocking agent: milk, BSA, serum, what percentage:5% milk in 1xTBST

Incubation time:1 hour

Incubation temperature:37 ℃

6. Primary Antibody

Species:

Reacts against:

At what dilution(s) have you tested this antibody:1:100 1:200 1:400 1:800

What dilution buffer was used: BSA

Incubation time: overnight

Incubation temperature: 4℃

What washing steps were done: wash 6 times (10min/time )

7. Secondary Antibody

Species:

Reacts against:

At what dilution(s) have you tested this antibody: 1:5000 1:10000

Incubation time: 1 hour

Wash steps: wash 6 times (10min/time )

Fluorochrome or enzyme conjugate: HRP

Do you know whether the problems you are experiencing come from the secondary? NO

8. Detection method
ECl, ECl+, other detection method: ECL (MILLIPORE)

9. Did you apply positive and negative controls along with the samples? Please specify.

10. Optimization attempts

How many times have you tried the Western? For 2 people to try

Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): no

Do you obtain the same results every time e.g. are background bands always in the same place? No band is exist

What steps have you altered? Concentration of primary antibody

Could you please help this customer to solve the problem?

Thanks for your kindly help

Best regards

ANSWER:

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear the customer has 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.

I would like to reassure you that ab6152 is tested and covered by our 6 month guarantee for use inWB and human samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

Reviewing this case, I would like to offer some suggestions to help optimize the results. I would also appreciate if you can confirm some further details:

1. Please confirm there is no staining at all? The blot is completely clear?

2. Please confirm if alysis buffer was used? I can recommend it would be important to lyse the cells in a proper lysis buffer such as RIPA if this has not already been tried, Sample buffer alone may not provide an adequate preparation of lysate, particularly as this is a nuclear protein.

3. After lysis, theLaemmli samplebuffercan beadded. The samples should be heated to 95oC 5 minutes to reduce and denature. I would suggest that 37oC will not be a high enough temperature to reduce and denature.

4. I recommend to try BSA rather than milk to block. Changing blocking agentcan sometimes help improve the results.

5. Is the current vial of secondary antibody working well with other primary antibodies? I would appreciate if you could provide further information regarding the secondary antibody. The supplier and product code?

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.

Many thanks for your quick response, the information was incredibly
useful. I have made a decision on the antibodies I would like to order
but was also wondering if you could send me some information on the
ELISA methodology used to validate them. Although the Applications
sections suggest the concentrations of antibody to use, it would be very
helpful to know more about the reagents, cell lines and precise
methods/kits used and whether they were validated as capture or
detection antibodies (or both).
The antibodies I am interested in are ab75717, ab1224 and ab6152. Any
notes you have on the validation of these antibodies would be greatly
appreciated.

ANSWER:

Thank you for your reply.

All three of these antibodies were validated using a protocol similar to the following:

http://www.abcam.com/index.html?pageconfig=resource&rid=11389

These antibodies were tested using BSA as a blocking agent. The antigen was recombinant or purified Cyclin D1, and the primary antibody dilution was in the range listed on each of their datasheets.

The ELISA assay will require optimization depending on your specific samples and experimental goals. The following reference is helpful for refining your protocol:

http://www.abcam.com/index.html?pageconfig=resource&rid=14665

While we have not validated these antibodies in a sandwich ELISA assay, it may be possible to use them for that purpose. As a starting point, you could consult our sELISA protocol:

http://www.abcam.com/index.html?pageconfig=resource&rid=11422

I hope this helps, please let me know if you need any other information.

I’m looking to set up an ELISA assay to monitor Cyclin D1 levels in immortalised PC3 cells. I am considering the antibodies available and need some advice on how best to decide on an antibody for use in a long term ELISA assay using MSD plates as well as clarify a few details on some of those available from AbCam.



Some general questions to start. Do the tested applications listed beside each antibody only refer to those in which the antibody was successfully tested? If ELISA is not listed, am I right to assume that the antibody was tested but failed in this particular application or not tested at all? Thus far, I have only considered antibodies with ELISA as a tested application, what makes an antibody more or less suitable for an ELISA?



I am currently looking at 3 antibodies; ab61758 (Rabbit polyclonal), ab75717 (Rabbit polyclonal) and ab6152 (Mouse monoclonal).



Firstly, the information on ab61758 refers to the immunogen it was raised against being a non-phosphopeptide. Does this mean the antibody will not detect the protein if it phosphorylated at this site? If this is indeed the case, would you not recommend the use of this antibody in a cell based system where Cyclin D could exist in both phosphorylated and non-phosphorylated states?



Secondly, would you recommend polyclonal antibodies for a long term assay given the inherent variability between experiments and batches? It is my understanding that this is less of an issue when raised against a smaller peptide region however I am unsure how small this peptide has to be (and indeed how small those used to raise ab75717 and ab61758 actually are) to be reasonably consistent and comparable to a monoclonal antibody.



And finally, are the more specific targets to which the antibodies are raised against (e.g. small peptide vs full length protein) generally regarded as more risky with if a folded protein structure is hiding the target in non-denaturing conditions?



I would greatly appreciate any advice you can offer to help me get started.

ANSWER:

Thank you for contacting us.
If we list an antibody as tested, that means the product has been validated for that application and we will cover it under our Abpromise guarantee. If we don't list an application as tested, it generally means that we do not have any data for that application. If we know that an antibody is unsuitable, there will generally be a note in the "Application Notes" section of the datasheet.
For ab61758, being raised against a non-phospho peptide means that the antibody will not detect Cyclin D when phosphorylated at threonine 286. If you are looking to detect both phosphorylated and non-phosphorylated forms, this antibody would not be suitable for that purpose.
Typically, when a polyclonal antibody is raised against an immunogen with a length of around 20 amino acids, they will provide the specificity and batch to batch stability of a monoclonal. Rabbit antibodies will also provide a higher binding affinity than mouse antibodies, so a rabbit polyclonal may be preferable if you are detecting a weakly expressed protein. The immunogen sequences for ab61758 and ab75717 are both very short and should provide the desired batch-to-batch stability.
If the antibodies are validated for IHC, as these are, that indicates that they are recognizing the native form of the protein. We will guarantee all three of these antibodies to recognize both native and denatured Cyclin D1.
I hope this helps, please let me know if you need any additional information.