Overview

  • Product name
  • Description
    Rabbit polyclonal to SEC61A
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Dog, Human, Pig
    Predicted to work with: Cow
  • Immunogen

    Synthetic peptide corresponding to Dog SEC61A aa 13-31.
    Sequence:

    CVILPEIQKPERKIQFKEK

  • Positive control
    • Pig microsomes

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    Preservative: 0.05% Sodium azide
  • Purity
    Whole antiserum
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab15575 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 1/10000. Detects a band of approximately 38 kDa (predicted molecular weight: 52 kDa).

Detects a band of approximately 38 kDa representing sec61 alpha from pig microsomes.

ICC/IF 1/1000. PubMed: 18784250

Target

Images

  • Anti-SEC61A antibody (ab15575) at 1/10000 dilution + Lysates prepared from pig microsomes

    Predicted band size: 52 kDa

References

This product has been referenced in:
  • Taverna E  et al. Non-canonical features of the Golgi apparatus in bipolar epithelial neural stem cells. Sci Rep 6:21206 (2016). IF . Read more (PubMed: 26879757) »
  • Buck TM  et al. The Lhs1/GRP170 chaperones facilitate the endoplasmic reticulum-associated degradation of the epithelial sodium channel. J Biol Chem 288:18366-80 (2013). WB . Read more (PubMed: 23645669) »
See all 5 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Question
Answer

DISCOUNT CODE: ***
Expiration date: February 16, 2013
Value: $337

This code will give you $337 off your next order before the expiration date. To redeem this offer, please submit an Abreview for ab15575 with drosophila samples and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: www.abcam.com/abtrial.

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Question

I will attempt to answer your questions below:
1. What's the species you were using to detect the proteins: human,
mouse, etc. proteins with a tag? HEK-293 lysates (human clones with
N-terminal V5-tag). Some abs from abcam (ab60332, ab80464) worked well.
2. Almost all of our antibodies are developed to work under denatured
conditions or otherwise it will be stated on the datasheet. It may be
that the antibody cannot detect its epitope as it may be simply not
accessible. Please boil your samples to provide optimal conditions.
The same goes for the reducing conditions of the gel which are
usually necessary for the most our antibodies to work properly.
I have tried denatured conditions are results were the same!
3. In order to avoid over-loading and therefore to decrease the side
bands, I would like to suggest loading 20 to 30 ug protein per lane.
I have loaded 20-40 ug protein per lane.
3. What are the listed primaries antibodies concentration? Or do you
use 1:5000 for all of them?
As you know, the primary antibody concentranctions vary, for instance:
ab15575 (1:5000), ab53532 (1:1000), ab96615 (1:1000) and ab106460 (1:1000)
4. What kind of secondary antibodies are you using for the listed
primaries? Do you perform a non-primary control with them beforehand?
depending on primary antibody used, secondary antibodies were: goat
anti-mouse IgGHRP (170-6516), goat anti-rabbit IgGHRP (170-6515) and
rabbit anti-goat IgGHRP (172-1034) from biorad.
5. Is it possible to send us an image?
6. How did you store the antibodies?
antibodies are stored as recommended, usually -20 C or sometimes 4C
P.s. note that I have already tested these antibodies at least 3 times
over the last 4 months. Hope to get replacement aliquots shortly.

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Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused.

As I have mentioned before, this two orders are way out of our Abpromise. However, I can see from the details you kindly provided the antibodies should have worked under these conditions, and I am therefore willing to make an exception in this case.


To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

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Question

Please find the WB questionnaire below:
1) Abcam product code ab: (ab15575, ab53532, ab96615 and ab106460)

3) Description of the problem: the mentioned antibodies are
non-specific i.e. several bands appear, none of which is the right size.
4) Sample preparation:
Type of sample (whole cell lysates, fraction, recombinant protein…):
cytoplasmic extract
Lysis buffer : Lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM
EDTA, 1% Nonidet P-40, 1 mM DTT and 10% glycerol) supplemented with
benzonase nuclease (250U, E1014, Sigma),
Protease inhibitors: 1 mM phenylmethylsulfonyl fluoride (P7626, Sigma)
and 1X protease inhibitor cocktail (Cat. No. 04693116001, Roche
Applied Science),
Phosphatase inhibitors
Reducing agent: 1 mM DTT
Boiling for ≥5 min? no
Protein loaded ug/lane or cells/lane: 60 ug
Positive control: V5-tagged constructs were detected with anti-V5 antibody
Negative control: empty cells
5) Percentage of gel: 10%
Type of membrane : nitrocellulose membrane
Protein transfer verified: by ponceau stain
Blocking agent and concentration: 5% Milk in TBS + 0.1% Tween 20
Blocking time: 2 h
Blocking temperature : RT
6) Was a fresh membrane used or had it been probed and stripped with a
different antibody? Yes
7) Primary antibody (If more than one was used, describe in
“additional notes”) : Mouse monoclonal anti-V5 (R960-25, Invitrogen)
Concentration or dilution: 1:5,000
Diluent buffer : 3% BSA in TBS
Incubation time: 16 h
Incubation temperature: 8 C
8) Secondary antibody: goat anti-mouse IgGHRP (biorad# 170-6516)
Species:
Reacts against: mouse
Concentration or dilution: 1:25,000
Diluent buffer : 3% BSA in TBS
Incubation time: 45 min
Incubation temperature: RT
Fluorochrome or enzyme conjugate:IgG-HRP
9) Washing after primary and secondary antibodies:
Buffer
Number of washes: 2
10)Detection method: autoradiography
11) How many times have you run this staining? 3
Do you obtain the same results every time? Yes
What steps have you altered to try and optimize the use of this antibody?
the anti-V5 antibody used has already been tested in the lab and found
to work well.
BR
Enzo
--
Enzo Scifo, PhD candidate
Meilahti Clinical Proteomics Core Facility
Institute of Biomedicine/Anatomy
P.O.Box 63 (Haartmaninkatu 8)
FI-00014 University of Helsinki
Helsinki, Finland
Tel: +358919125202
Fax: +358919125206
Mobile: +358466370309

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Answer

Thank you for taking time to complete our questionnaire. I am sorry to hear that 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 can confirm that unfortunately, as you purchased this product farover 180 days ago it is will no longer be covered by our Abpromise guarantee on this occasion. Details of our guarantee are on our website and we encourage customers to contact us as soon as possible if they experience any difficulties.If a particular antibody causes problems we do encourage customers to contact us as soon as possible to save time and material. Although not covered by our guarantee, we would still be pleased to offer some suggestions to help optimize the results

Having reviewed this case, I would like to offer some suggestions to help optimize the resultsandI would also appreciate if you can confirm some further details:

1. What's the species you were using to detect the proteins: human, mouse,etc. proteins with a tag?

2. Almost all of our antibodies are developed to work under denatured conditions or otherwise it will be stated on the datasheet. It may be that the antibody cannot detect itsepitope as it may be simply not accessible. Please boil your samples to provide optimal conditions. The same goes for the reducing conditions of the gel which are usually necessary forthe most our antibodies to work properly.

3.In order to avoid over-loading and therefore to decrease the side bands, I would like to suggest loading 20 to 30 ug protein per lane.

3. What arethe listedprimaries antibodies concentration?Or do you use 1:5000 for all of them?

4. What kind of secondary antibodies are you using for the listed primaries? Do you perform a non-primary control with them beforehand?

5. Is it possible to send us an image?

6. How did you store the antibodies?


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


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

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Answer

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.


If you wouldn't mind, I would like to try to understand what may be contributing to the problems encountered in order to hopefully resolve the situation. To this end, could you please fill in the questionnaire I have attached to this email. If you could include images of the results obtained (including that using the V5 antibody) that would be very helpful.

I also have a few additional questions:

1. How were the V5-protein construct made? Which sequences were used to construct the fusion protein (human/mouse/rat etc)?

2. Which V5 antibody was used? And which secondary antibody was used with this antibody?

3. From your orders I can see that you also purchased ab80464, ab60332 and ab84589, were you using the same protocol using these (V5 tagging?)? Did you have any problems in using these antibodies as well?

OnceIhavereceive this information and the completed questionnaire,I will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary.

I look forward to receiving your reply.

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Answer

Thank you very much for your inquiry. Unfortunately we do not have an antibody in our catalogue for the moment for which we can suggest a crossreactivity with the yeast SEC61 proteins. I am very sorry for this. Please see further below for the alignment scores. For the TOMM22 protein, I could not find the sequence in UniProt neither. Can the customer maybe provide this? In the case we can not make an alignment, I am willing to provide a testing discount. alignment scores for SEC61 antibodies: ab15575 44% yeast/ 61% schp http://www.ebi.ac.uk/Tools/services/web_clustalw2/toolresult.ebi?tool=clustalw2&jobId=clustalw2-I20110905-173253-0036-74194366-pg ab77789 and ab15037 14% yeast/schp http://www.ebi.ac.uk/Tools/services/web_clustalw2/toolresult.ebi?tool=clustalw2&jobId=clustalw2-I20110905-173532-0061-46398952-pg ab97537 15% yeast/schp http://www.ebi.ac.uk/Tools/services/web_clustalw2/toolresult.ebi?tool=clustalw2&jobId=clustalw2-I20110905-173733-0295-24017780-pg ab1327 14% yeast/schp http://www.ebi.ac.uk/Tools/services/web_clustalw2/toolresult.ebi?tool=clustalw2&jobId=clustalw2-I20110905-173823-0394-68897710-pg Unfortunately I have not found the SEC61B and G yeast sequences on UniProt and can therefore not provide an alignment. Do you have these sequences? I am sorry for not being able to be more helpful on this occasion. Please provide me with the sequences if you can in order for me to do the alignments. Thank you very much.

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