Anti-GM130 antibody (ab31561)

ok donc il faudra que je revienne vers vous pour l'obtenir. A priori je vais le tester en WBlot d'abord et puis ensuite en Immuno-fluorescence.

ANSWER:

Je recommanderais un desdeux anti-GM130 suivants :

ab103416 (testé en Western Blot et IHC-P)dont l'immunogèneprésente 87% d'homologie avec la protéine du xénope (voir pièce jointe), http://www.abcam.com/ab103416

ab31561 (testé en Western Blot) dont l'immunogène présente 93% d'homologie avec la protéine du xénope(voir pièce jointe), http://www.abcam.com/ab31561



Merci dem'indiquer quel anticorps vous souhaitez tester et je vous enverrai le code pour l'offre de test.

Voici la séquence de GM130 de xénope (elle provient du site xenbase) en format word.
J'ai une question: est ce qu'il faut que je précise un code d'offre ou quelque chose d'autre sur le bon de commande?

ANSWER:

Merci pour la séquenceGM130 de Xénope.

Vous n'avez pas à indiquer de code sur la commande originale de l'anticorps que vous voulez tester. Vous indiquerez le code promotionnel :
1) lors de l'envoi de vos résultats sous forme d'Abreview
2) sur le bon de commande de l'anticorps gratuit.

Afin de vous conseiller l'anti-GM130 le mieux adapté à vos travaux, pourriez-vous m'indiquersi vous l'utiliserez en Western Blot, en IHC-P, ou dans autre application?

Merci.

1) Abcam product code ab
Ab31561
2) Abcam order reference number or product batch number
3) Description of the problem
Ab31561 is anti-Golgi antibody and suppose to stain Golgi apparatus. Yet when use in IF, stain everywhere in the cell (non specific staining)
4) Sample preparation:
Species Hela cell was fixed with 3.7% formaldehyde solution for 15min
Type of sample: Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed paraffin embedded sections, cells in culture, other:
Sample preparation
Positive control
Negative control
5) Fixation step
Yes/No yes
If yes: Fixative agent and concentration 3.7% formaldehyde solution in PBS
Fixation time 15min
Fixation temperature room temperature
6) Antigen retrieval method (if used)
7) Permeabilization method: 0.2% Triton X-100 in PBS 5-10min at room temperature
Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers?
Permeabilizing agent and concentration:

8) Blocking agent (eg BSA, serum…): 3% milk
Concentration 3%
Blocking time 30min
Blocking temperature room temperature (also try 4 degree ON)
9) Endogenous peroxidases blocked?
Endogenous biotins blocked?
10) Primary antibody (If more than one was used, describe in “additional notes”) : Ab31561
Concentration or dilution 1:200 (also try 1:500)
Diluent buffer 3%milk
Incubation time 30min at RT
11) Secondary antibody: Texas Red goat anti rabbit IgG (work well in other experiment)
Species:
Reacts against:
Concentration or dilution 1:200
Diluent buffer 3%milk
Incubation time 30min at RT
Fluorochrome or enzyme conjugate
12) Washing after primary and secondary antibodies:
Buffer 3%milk
Number of washes 3 times (3min each time)
13) Detection method
Fluorescence microscope
14) How many times have you run this staining? 4
Do you obtain the same results every time? yes
What steps have you altered to try and optimize the use of this antibody? Try different dilution (1:200 1:500) incubate at different condition (RT 30min/4 degree ON)

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.

Having reviewed this case, I would like to offer some suggestions to help optimise the results from ab88530 in ICC :

I would suggest to try a fixation of the cells using ice cold acetone or methanol. PFA may mask the antigen.
I would also recommend to use another blocking agent : 1% BSA in PBST or 10% serum from the species in which the secondary antibody was raised.
Did you run a "no primary" control to determine if the non-specificity was due to the secondary antibody?



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.

Our lab ordered your Anti-GM130 antibody (ab31561) one month ago. Recently, we try this antibody in Immunofluorescence assay. According to the photo on your websites. It works well in the Immunofluorescence assay. However, follow the instruction on your websites, our results show the antibody just stain everywhere in the cell.
Attached are the image of our Immunofluorescence results. I wonder if there are something wrong with the antibody. Also, I wonder if you could send us the right antibody which specifically stain Golgi apparatus.
Thanks.

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.

I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we 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. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.

I look forward to receiving your reply.