Question (48038) | HSD11B1 peptide (ab101097)

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Question

Sir/Madam,

We have bougthHSD11B1 antibody (ab39364) from your Company in December 2010. We have tried this antibody on microsomes from rat liver and have obtained very interesting results: on the same samples of liver microsomes one time we got a double band on approximatelly 34 kDa and one band on 43 kDa (Picture 1, A), while other time on the same samples we obtained one band on 34 kDa and bands on app. 53 and 43 kDa (Picture 2, A).At the sametime on microsomes from rat adipose tissue we got one bend on 34 kDa (picture 3). Since we were not assure about specificity of our bands in liver samples, we bought a blocking peptide for this antibody (ab101097) from Institute for Nuclear Sciences VINCAthrough PIU tender. We prepared experiment with blocking peptide as you suggested in your protocol (BLOCKING WITH IMMUNIZING PEPTIDE (BL) PROTOCOL) using final concentration of HSD1 antibody 1 microgr/ml and blocking peptide 5 microgr/ml (5 fold excess as you suggested in peptide competition assay). In the first experiment (Picture 1, B) using of blocking peptide led to disappearance all bends, while in other experiment (Picture 2, B) specific band on 34 kDa showed faint signal while other bands totally dissapeared.

Questions:

1. Does HSD1 antibody (ab39364) recognize HSD2, since it is on 44 kDa and due to high similarity between HSD1 and HSD2?

2. Is the protocol with blocking peptide correct, should I use lesser concentration of blocking peptide (for example 2 fold excess) to obtain better results?

3. In case od double bands on 34 kDa, which band I should use as specific?

As evidence I enclosed scan images ofthree different Western blot membranes probed with this antibody.

Thank you in advance forquick answer.

Best regards,

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 would like to reassure you that this antibody is tested and covered by our 6 month guarantee forWB and forMouse, Rat andHuman samples. 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.

In answer to your questions:

1. Does HSD1 antibody (ab39364) recognize HSD2, since it is on 44 kDa and due to high similarity between HSD1 and HSD2?

I can confirm we are not aware of any data to suggest that this antibody would show cross reactivity to recognize HSD2.

2. Is the protocol with blocking peptide correct, should I use lesser concentration of blocking peptide (for example 2 fold excess) to obtain better results?

I can suggest to consider using a higher concentration of blocking peptide as the blockingmay not becomplete.

You mention that in the first experiment use of blocking peptide led to disappearance all bands, while in other experiment at 34 kDa there is a faint signal. I am sorry I do not know how to explain this difference between two uses of the same antibody and the same blocking peptide, except that this may indicate incomplete blocking.

3. In case of double bands on 34 kDa, which band I should use as specific?

This would be difficult to answer. As the results show nonspecific bands,in order to identify those bands in the rat liver microsome samples further experiments would be necessary. Using a higher concentration of blocking peptide may help in this case so you can observe which of the bands remains.

This antibody ab39364 has been confirmed to be cross reactive with rat 11β-HSD1 and the expected band should appear at 32 kDa. Unfortunately, I also did not find anything in literature to suggest there are any different size isoforms of SD11B1 in liver samples. You may like to try a more extensive literature search to confirm this.

I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

I would also appreciate if you could confirm: if the same samples used in both experiments?

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



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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