Anti-58K Golgi protein antibody - Golgi Marker (ab5820)

Here our immunocytochemistry protocol is.
Best regards,
1) Abcam product code ab5820
2) Abcam order reference number or product batch number lot 957678
3) Description of the problem
The staining did not show perinuclear distribution as expected for the Golgi.
4) Sample preparation:
Species HeLa cells
Type of sample: PFA fixed
Sample preparation
Positive control: none
Negative control: the staining was done only with secondary antibody
5) Fixation step
Yes
If yes: Fixative agent and concentration 4% PFA Fixation time: 10
minutes Fixation temperature: room temperature
6) Antigen retrieval method was not used
7) Permeabilization method:
Did you do a permeabilization step (details please) or add
permeabilizing agent in any dilution buffers?
Permeabilizing agent and concentration:
Permeabilization of cells was done with PBS containing 0.1% Triton
X-100 and 0.2% BSA with following incubation for 15 minutes at room
temperature.
8) Blocking agent (eg BSA, serum?): BSA, goat serum
Concentration: 2% BSA, 5% goat serum
Blocking time: 1 hour
Blocking temperature: room temperature
9) Endogenous peroxidases blocked? no
Endogenous biotins blocked? no
10) Primary antibody (If more than one was used, describe in
"additional notes"): Anti-58K Golgi protein antibody - Golgi Marker
Concentration or dilution: 1:500 Diluent buffer: PBS containing 0.1%
Triton X-100 and 5% goat serum Incubation time: overnight at 4C
11) Secondary antibody: Alexa Fluor 568 goat anti-rabbit IgG
("Invitrogen")
Species: Goat
Reacts against: Rabbit
Concentration or dilution: 1:400
Diluent buffer: PBS containing 0.1% Triton X-100 and 5% goat serum
Incubation time: 1 hour at room temperature Fluorochrome or enzyme
conjugate: orange-red-fluorescent Alexa Fluor 568 dye
12) Washing after primary and secondary antibodies: Buffer PBS
Number of washes 3 times for 3 minutes
13) Detection method: Fluorescence visualization was done using Leica
SP2 confocal microscope
14) How many times have you run this staining? 2
Do you obtain the same results every time? Yes
What steps have you altered to try and optimize the use of this
antibody? Different dilutions of antibody have been tested.
Document attachment: Attaching images of your IHC is strongly
recommended and can greatly speed up our investigation of your problem.

ANSWER:

Thank you for sending us some further details of the protocol.
My colleague is out of office and she has asked me to look after her customers in her absence.
The protocol looks fine to me and I have only a few things to suggest which may improve the signal:
1) Cell culture:
It may well be that the cells are not entirely healthy; some signs of apoptotic "blebbes" can be seen. It would be worth plating the cells onto the slides/coverslips 24-48 hrs before the experiments. The cells should be in the exponential growth phase and it would be important to make sure that they are well fed. If it is possible, please use the high passage numbers since after 12-15 passages, the cells tend to undergo serious changes and lose their "morphology".
2) Fixatives:
Have you tried different fixatives to see if the appearance of the signal or the "artifacts" changes. For this purpose, you could test ice-cold methanol/ethanol/acetone.
I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.

We have had serious problems with the golgi marker we bought from you. We have used PFA fixed HELA cells and surprisingly the staining does not look at all like it should, see the file attached. We have tried several dilutions also. Do you have suggestions what to do?
with best regards

ANSWER:

Thank you for contacting us and sending the image.
Without further details it is very difficult to make suggestions to improve the results from this antibody.
I will be very happy to help you, and for that, I would appreciate if you could please send me more protocol details. I send you a questionnaire that only take about 5 minutes to be completed, and could help us understand the results obtained.
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. If no suggestions can be made to the protocol, and the antibody was purchased within the last 6 months, it is covered by our Abpromise guarantee, and therefore I can offer you a free of charge replacement or a credit note for it.
I look forward to receiving your reply. Please do not hesitate to contact us if you need any more advice or information.

WB : no band.
HeLa cells.
different dilutions up to 1/200
incubation 2h RT and O/N 4ºC
5% milk.

ANSWER:

Merci de nous avoir contactés.

Nous sommes désolés d'apprendre que le produit ab5820 que vous avez reçu ne fonctionne pas comme attendu.
Comme convenu j'ai mis en place l'envoi d'une unité de ab27043. Le numéro de commande de remplacement gratuit est : ******. Vous recevrez prochainement un mail de confirmation comprenant les détails d'expédition.

N'hésitez pas à nous contacter lors d'une prochaine occasion.

WB with rat samples
looking for Golgi marker proteins - antibodies and pos. controls
sent link to list of golgi marker proteins

ANSWER:

Thank you for contacting us.

This is the link I found and which gives you a list of Golgi marker proteins including references and a brief description.

http://www.antibodybeyond.com/reviews/organelle-markers/golgi-marker.htm

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Como te decía en el otro e-mail nosotros no conseguimos una única banda cuando trabajamos con el anticuerpo de golgi conseguimos 3. He rellenado el cuestionario que me has mandado, te he adjuntado también una imagen de los blot que consigo. Gracias

ANSWER:

Muchas gracias por enviarnos el cuestionario relleno y la imagen del blot. Lamento muchísimo que el anticuerpo no os haya dado buenos resultados, y después de haber estudiado el protocolo enviado me gustaría haceros algunas sugerencias para intentar mejorarlos. En caso de que no se consiga obtener el resultado esperado del anticuerpo, al estar éste aun en garantía puesto que se adquirió hace menos de 6 meses, estaré encantada de mandaros un reemplazo del mismo (de un lote diferente, o bien de otro anticuerpo si lo hubiera disponible contra la misma proteína), devolveros el dinero, o haceros un vale sin caducidad para una futura compra. Para intentar obtener tan solo la banda correspondiente a la proteína de interés, yo sugeriría las siguientes pautas: -Usar buffer RIPA para lisar las células, es el que solemos usar en el laboratorio y nos da buenos resultados para detectar proteínas desnaturalizadas. -Disminuir la concentración tanto de anticuerpo primario, como de secundario. Os recomiendo, si podéis, correr varias diluciones más concentradas en paralelo, y observar si mejoran los resultados. Para el primario 1/2000 puede ser un buen punto de partida, y 1/8000 para el secundario. -Nosotros siempre recomendamos usar controles positivos. En este caso ayudaría a dilucidar cuál de las bandas corresponde a la proteína de interés, y descartar fallo del anticuerpo. Para este caso concreto se recomienda usar hígado (de rata, ratón, humano…). Tenemos varios lisados celulares procedentes de hígado si lo requirieseis para usar como control positivo. -También es importante, en casos en los que se obtienen varias bandas, llevar a cabo un control no primario. Es decir, incubar el anticuerpo secundario solo, sin primario, y comprobar que no generan ninguna banda en la membrana. Si después de llevar a cabo estos intentos de mejora seguís sin obtener los resultados esperados, otra cosa que podríais probar es incubar el primario con el péptido de bloqueo, que está disponible en nuestro catálogo, y os lo podríamos mandar como reemplazo del anticuerpo primario. http://www.abcam.com/index.html?datasheet=13733 Espero haberos ayudado. Si necesitáis cualquier otra información o ayuda, por favor, no dudéis en contactarnos de nuevo, y desde luego, si el anticuerpo sigue sin funcionar como debiera, te ruego que nos vuelvas a escribir para poder solucionar este problema lo más rápido y eficazmente posible.