Anti-Sarcomeric Alpha Actinin antibody [EA-53] (ab9465)

What percentage of sodium azide is in the buffer of this antibody?

ANSWER:

Thank you for your call yesterday and for your patience while I've looked into your enquiry.

I've heard back from the lab, and there is 0.05% sodium azide in this antibody. I will update the datasheet and have an MSDS madepromptly. Please let me know if you have any further questions or if there is anything else that we can do for you, and I'll be happy to help.


Thank you for help about this case.
Attached please find the detail and data.
Would you please help deal with this issue?

Thank you very much.
Best regards,

Iris

1) Abcam product code ab9465

2) Abcam order reference number or product batch number GR50514-3

3) Description of the problem
1. ICC for cardiomyocytes, striated form was not appeared.
2. Non-specific binding for 3T3 fibroblasts.
4) Sample preparation:
Species mouse
Type of sample: Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed paraffin embedded sections, cells in culture, other: cells in culture (cardiomyocytes primary culture/ 3T3 fibroblasts)
Sample preparation
Positive control
Negative control

5) Fixation step
Yes/No Yes
If yes: Fixative agent and concentration 3.7% PFA in PBS
Fixation time 20 min
Fixation temperature 4℃
6) Antigen retrieval method No

7) Permeabilization method:
Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers? Yes, 15 min, room temp.
Permeabilizing agent and concentration: 0.1% Triton X-100 in PBS

8) Blocking agent (eg BSA, serum…):
Concentration 2%
Blocking time 20 min
Blocking temperature room temp.

9) Endogenous peroxidases blocked? no
Endogenous biotins blocked? no
10) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution 1:100
Diluent buffer 2% BSA in 0.1% PBST
Incubation time 1 hr room temp.


11) Secondary antibody:
Species: rabbit
Reacts against: Mouse
Concentration or dilution 1 hr
Diluent buffer 2% BSA in 0.1% PBST
Incubation time Fluorochrome or enzyme conjugate FITC

12) Washing after primary and secondary antibodies:
Buffer PBS,
Number of washes three˜five times, 5 mins
13) Detection method microscope

14) How many times have you run this staining? 5 times
Do you obtain the same results every time? Yes
What steps have you altered to try and optimize the use of this antibody? No

ANSWER:

Thank you for taking time to complete our questionnaire and for contacting us. I am sorry to hear this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.

Having reviewed the protocol details, I believe this product should have given satisfactory results. It appears that you may have received a faulty vial.

I apologize for the inconvenience and am pleased to offer you a free of charge replacement or credit note in compensation.

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

I could finally make this staining work by using an enzymatic (protease-based) antigen retrieval method instead of citrate. Dilution 1:800 looks ideal in this condition.
Thanks for your help,
Regards,

ANSWER:

Thank you for taking the time to contact me and let me know.

I am very pleased to hear the staining is now working with the enzymatic retrieval. This is also useful information for us regarding this product and has gone into our quality monitoring records.

Good luck with your future work and if you have any further questions, please do not hesitate to contact me.

Dear Sir or Madam,

I am trying to troubleshoot IHC-P staining (ABC method) of infarcted pig heart with your actinin antibody. I've tried several antibody concentrations and antigen retrieval methods (citrate 10 min in pressure cooker or 20 min waterbath) but haven’t been convinced of my results so far. Indeed, I can observe a nuclear staining as well as a vessel staining (which should not be stained because they express smooth muscle actinin, which your antibody should not recognize). I therefore think my staining is mostly aspecific. I think the main issue is the antigen retrieval method. Which antigen retrieval method would you recommend (method, time, temperature)? I could also increase the incubation time with the primary antibody… Would you have other suggestions?

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 thatin 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.

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. This will provide us with further information, including details of the specific antibody you are using (please provide the product code), and I hope I will then be able to provide some further suggestions to you.

I would appreciate if you are also able to provide an image which would help us to assess the results.

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

Order Details
Antibody code:

Lot number:

Purchase order number
or preferably Abcam order number:


General Information
Antibody storage conditions (temperature/reconstitution etc)


Description of the problem (high background, low signal, non-specific staining etc.)

Sample (Species/Tissue/Cell Type/Cell Line etc.)


Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)


Antigen retrieval (Enzymatic method, Heat mediated technique etc.)


Permeabilization step


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


Positive and negative controls used (please specify)


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



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

Do you obtain the same results every time?
Yes No


What steps have you altered?


Additional Notes


We would appreciate if you are also able to provide and image which would help us to assess the results

Hi, I am interested in purchasing antibodies for positive staining of human cardiomyocytes grown in cell culture. The antibodies recommended were sarcomeric alpha actinin (+ve), slow muscle myosin (+ve) and CD90 (-ve). We were planning on staining the whole cells using an immunofluorescence or confocal microscope. Could you please advise on which antibodies to purchase (primary and secondary) and indicate the protocol which would be most useful for whole cell staining. Many thanks,

ANSWER:

Thank you for your enquiry and your interest in our products.
I have conducted a search for you and found several antibodies against Sarcomeric Alpha Actinin. One of them is ab9465 which has been tested in ICC/IF and it recognizes cardiac muscle in different species (Mouse, Rat, Sheep, Rabbit, Goat, Chicken, Hamster, Cow, Cat, Dog, Pig, Xenopus laevis, Fish, Lizard, Snake, Xenopus tropicalis ). We have excellent feedback (3 Abreviews) from our customers who have used it and also specific publications (11). As you can see on the databases, it is a mouse monoclonal antibody and the isotype is IgG1.
For selecting the correct compatible secondary antibodies, I would advise you to use our advanced search engine: http://www.abcam.com/index.html?pageconfig=productmap&cl=918
You can type the target and the host species, isotype, format, specificity, conjugate etc. Have you considered the fluorochromes you wish to use?
Here are a few suggestions:
ab97239: Goat anti-Mouse IgG1 heavy chain (FITC) secondary antibody,
ab98692: Goat polyclonal Secondary Antibody to Mouse IgG1 - heavy chain (FITC), pre-adsorbed
ab72553: Goat polyclonal Secondary Antibody to Mouse IgG1 (SureLight® Allophycocyanin)
Regarding ICC protocol, I would advise you to take a look at the Protocol section at our website. We have a great numbers of general protocols which may be useful for you:
http://www.abcam.com/index.html?pageconfig=popular_protocols
http://www.abcam.com/index.html?pageconfig=resource&rid=11417
http://www.abcam.com/index.html?pageconfig=resource&rid=12129
I hope this helps and if I can assist further, please do not hesitate to contact me.