Overview

  • Product name

  • Description

    Rabbit polyclonal to DAAM1
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WBmore details
  • Species reactivity

    Reacts with: Human
    Predicted to work with: Mouse, Rat, Rabbit, Horse, Chicken, Guinea pig, Cow, Cat, Dog, Zebrafish
  • Immunogen

    Synthetic peptide corresponding to a region within internal sequence amino acids 396-445 (GNTVQYWLLL DRIIQQIVIQ NDKGQDPDST PLENFNIKNV VRMLVNENEV) of human DAAM1 (NP_055807).

  • Positive control

    • 293T cell lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab84526 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB Use a concentration of 1 µg/ml. Predicted molecular weight: 123 kDa. Good results were obtained when blocked with 5% non-fat dry milk in 0.05% PBS-T.

Target

  • Relevance

    DAAM1 (Dishevelled associated activator of morphogenesis 1) is a diaphanous-related formin which binds to dishevelled (Dvl) and Rho, and mediates Wnt-induced Dvl-Rho complex assembly. It is an essential component of noncanonical Wnt signaling. DAAM1 also communicates with Rho GTPases, CIP4 and Src in the regulation of the signalling pathways that co-ordinate the dynamics of the actin filament system. DAAM1 may be a coordinator of endocytosis and cytoskeletal remodeling.
  • Cellular localization

    Cytoplasmic. Note: Perinuclear.
  • Database links

  • Alternative names

    • DAAM 1 antibody
    • Dishevelled associated activator of morphogenesis 1 antibody
    • FLJ41657 antibody
    • KIAA0666 antibody

Images

  • Anti-DAAM1 antibody (ab84526) at 1 µg/ml (in 5% skim milk / PBS buffer) + 293T cell lysate at 10 µg

    Secondary
    HRP conjugated anti-Rabbit IgG at 1/50000 dilution

    Predicted band size: 123 kDa
    Observed band size: 144 kDa
    why is the actual band size different from the predicted?



    Gel concentration: 6-18%

References

ab84526 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Answer

Thank you for your reply and sorry for the delay in mine.

I think the band at ˜150 kDa could well be the expected band. You could see this clearer by performing your experiment using the siRNA experiment you carried out.

However, I can understand your concern. If you would like I can offer you a replacement antibody to try such as the mouse monoclonal ab56951 or the rabbit polyclonal ab71327 or ab71499. Alternatively, if you would prefer, a credit note or refund.

I look forward to hearing how you would like to proceed.

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Answer

Thank you for your reply and sharing your results with me.

It seems from the image that the BSA blocking has been much more successful. Although the band at ˜50 kDa has remained, there is now a clear band at around the expected 150 kDa. This is shown very clearly in your U2OS cell line but also in the MDCK cell line.

The no-primary control does suggest there is quite a bit of background generated from the non-specific binding of the secondary antibody to the membrane. In order to reduce this as well as the non-specific band at ˜50 kDa, I would consider the following:

1. Drying a higher dilution of the secondary antibody, maybe try 1/20,000.

2. Increasing the level of Tween in the buffers to 0.1%.

3. After each incubation include a wash step (3x5 minutes) with TBS with tween 0.1%, unless you are already performing this?

4. Did you incubate the primary antibody overnight at 4°C as suggested?

5. The results may improve if less protein is loaded on the gel, try 5 or 10 ug per well?

6. You could now consider reducing the concentration of primary antibody used, I would try 1/250 or 1/500.

I hope this information has been of help. If I can be of any further assistance, please do let me know.

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Question
Answer

Thank you for taking time to provide that extra information. It has made understanding what may be contributing to the problems observed much easier to ascertain.

From the results shared of the blot performed with the xxxxxx antibody it appears that a specific band can be seen at ˜130 kDa . However, as with the ab84526 there appears to be strong non-specific band at ˜60 kDa. There are a few things I would consider changing in your protocol which may improve the results of both the antibodies:

1. I would suggest using a lysis buffer such as RIPA (recipe below). This can aid in the more complete lysis of the sample:

150 mM sodium chloride

1.0% NP-40 or Triton X-100

0.5% sodium deoxycholate

0.1% SDS (sodium dodecyl sulphate)

50 mM Tris, pH 8.0

2. I noticed you are using both milk and BSA in your blocking buffer. We have found that this can sometimes lead to high background in Western blotting. I would therefore suggest trying one blot with 5% milk blocking and another with 5% BSA blocking and seeing which produces the best results. I would increase to 0.1% Tween 20 in your buffers.

3. I would also suggest introducing blocking into the antibody diluents, 1% of either milk or BSA, but make sure to use the same as was used in the blocking step. We also find that more specific binding can be achieved by incubating the primary antibody with the blot overnight at 4°C instead of 1 hour at room temperature as you are currently using.

4. For both the xxxxx and ab84526 I think it would be worthwhile to try a "no primary" control. Just to check that the non-specific band at ˜60 kDa is not from the secondary antibody non-specifically binding to the blot.

Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

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Answer

Thank you for your reply and sorry for the delay in getting back to you.

xxxx is currently away from the office but I will try to help as best I can in this case. The information you have provided in regards to the experiment you have carried out has been very helpful in understanding what may be contributing to the problems observed. I'd like to clarify a few further points if possible. This will hopefully allow us to ascertain the likely cause of the problems and resolve them. Therefore, would you mind answering the following questions:

1. You mentioned using cell lysis buffer to lyse the cells. Could you let me know which kind of buffer (the components) were used for this?

2. Could you let me know which kind of blocking buffer was used and at what concentration? (milk, BSA?)

3. What buffer was used to dilute the primary and secondary antibodies?

4. What buffer was used for the wash steps?

5. You have mentioned that a different antibody worked very well with your samples. Could you let me know which antibody this was?

6. The secondary antibody used, has it been used successfully before with other antibodies?

7. You have mentioned that an alternative anti-DAAM1 antibody from proteintech was used, was this with the same U2OS samples? Could you share an image of the results obtained?

8. The protein DAAM1 is quite large, was the transfer from the gel to the membrane assessed? By using something like Ponceau red staining? Or did the protein ladder transfer as expected?

I am sorry for all the questions but I hope to be able to refine what may be contributing to the problems encountered.

Could you finally also confirm that I have the correct order information for you.

Order number xxxxx (purchase order number xxxxxxx) delivered around the 26th of October?

I look forward to receiving your reply.

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Answer

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

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been succesful.

I would like to reassure you that this antibody is tested and covered by our guarantee for WB and human samples. Before deciding how to proceed, I would like to investigate this particular case further for you, and also obtain some further information for our quality records.

In order to do this, I have enclosed a questionnaire below to otbtain further information regarding the western blot. I would appreciate if you could complete this. It will help you put the information we require together very easily.

In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

Thank you for your time and cooperation. We look forward to receiving the completed questionaire.



Order Details
Antibody code:

Problem
Choose: Non-specific band Multiple bands No signal or weak signal High background


Lot number

Purchase order number
or preferably Abcam order number:



General Information
Antibody storage conditions (temperature/reconstitution etc)


Description of the problem (high background, wrong band size, more bands, no band etc.)


Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)


Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)


Amount of protein loaded


Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)


Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method (ECL, ECLPlus etc.)


Positive and negative controls used (please specify)



Optimization attempts (problem solving)
How many times have you tried the Western?



Have you run a "No Primary" control?
Yes No

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


What steps have you altered?


Additional Notes:


Image:
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.

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