Anti-Somatostatin Receptor 1 antibody (ab2366)

Buenas tardes,
Recientemente adquirimos el anticuerpo contra receptor de somatostatina 1: Ab2366
Queremos poner a punto un protocolo de Western Blot, con células humanas y de rata, incluyendo control positivo de línea pancreática (PANC-1)
Tienen información sobre la dilución óptima de este anticuerpo para un protocolo de Western? revelamos con Fosfatasa alcalina.
Muchas gracias y saludos

ANSWER:

Gracias por contactarnos.

La concentración de Ig de este anticuerpo es de 66.6ug/ml. Las diluciones óptimas varían en el rango entre 1:40 y 1:400.

Si necesitas consultar nuestros protocolos online, te adjunto el link a la página:

http://www.abcam.com/index.html?pageconfig=popular_protocols

Espero haberte ayudado. Para más información, no dudes contactarme de nuevo.

1) Abcam product code ab 2366 (anti SST1 )

2) Abcam order reference number or product batch number

3) Description of the problem aspecific reattivity?! Does it work?

4) Sample preparation:
Species
Type of sample: Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed paraffin embedded sections, cells in culture, other:
Sample preparation
prostate and colon cancers, cell lines from colon ca (WIDR,LOVO, LRWZ), and from breast cancers (MCF7, MDAMB231)


Negative control Ab omission in the same tissues

5) Fixation step formaline 10% 12-24 h
Yes/No
If yes: Fixative agent and concentration
Fixation time
Fixation temperature r.t

6) Antigen retrieval method citrate buffer 30 min and or EDTA

7) Permeabilization method:
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…):
Diluent Dako used with primary AB at 1:400 or 1:500 for 30 min or 1 h
Concentration
Blocking time
Blocking temperature

9) Endogenous peroxidases blocked? Hydrogen peroxide (H2O2) 10 min
Endogenous biotins blocked? Yes

10) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution 1:60
Diluent buffer Dako
Incubation time 60 min

11) Secondary antibody: Streptavidine –Biotin (Dako)
Species:
Reacts against:
Concentration or dilution
Diluent buffer
Incubation time
Fluorochrome or enzyme conjugate Dab 10 min

12) Washing after primary and secondary antibodies: PBS 1X (2 times for 5 min)
Buffer
Number of washes

13) Detection method

14) How many times have you run this staining? 3 times
Do you obtain the same results every time?
What steps have you altered to try and optimize the use of this antibody? We tested different Ab dilution and another antigen retrieval buffer (EDTA at 98,5°C for 20 min)

Document attachment: Attaching images of your IHC is strongly recommended and can greatly speed up our investigation of your problem.


In particular we tested Anti SST1 Antibody with positivity?! mainly In the cytoplasm and in the nucleus ?! of cells. Could you help me? I expected positivity in the cell membrane.
Thank you very much

ANSWER:

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality. I would like to reassure you that this antibody is tested and covered by our 6 monthguarantee forIHC-P, ICCand human samples.

Reviewing this case, I would like to offer some suggestions to help optimize the results from ab2366. I would also appreciate if you can confirm some further details:

1. Please provide the Abcam order number and date of purchase.

2. Could you confirm there is background staining? I would appreciate if you are able to provide some images (from both the IHC-P and ICC) which would help us to assess the results.

3. For ICC staining of thecell lines, I can recommend to fix in 4% PFA for 10 minutes only. No antigen retrieval isthen required.

(For the IHC-P on tissue sections, I would recommend to continue with the fixation and antigen retrieval you are already using).

4. To help reduce the background staining, I can suggest to use a blocking step, such as 10% serum for 20 - 30 minutes if you have not already done so.

5. I can recommend to try a lower concentration of antibody to reduce the background. Try 1:200 or even 1:500. Incubating overnight at4oC can often provide more efficient and specific staining.

6. Could you confirm ifthe current vial of secondary antibody isworking well with other primary antibodies? I can recommend to consider including a no primary control in order to assess if this may be binding non specifically. The concentration of the secondary may need to be reduced in order to optimize the results.

7. I can suggest to include0.2% Tween in the wash buffer. This would help to wash away any excess antibody.

In the event that a product is not functioning in the applications 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 hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

BATCH NUMBER 231735 ORDER NUMBER c568472

DESCRIPTION OF THE PROBLEM No staining, all cells had non-specific staining.

SAMPLE Mouse cells which are pituitary somatotropes FACS sorted and fixed in wells of 96-well plate.

PRIMARY ANTIBODY AB2366 somatostatin receptor rabbit polyclonal to mouse. Used at 1/50 diluting with blocker left overnight at 4C. Next day wahed with PBS for 45 min.

DETECTION METHOD Looked at cells in PBS on Phase contrast microscope.

POSITIVE AND NEGATIVE CONTROLS USED Secondary alone antibody.

ANTIBODY STORAGE CONDITIONS 4C

FIXATION OF SAMPLE 4% PFA

ANTIGEN RETRIEVAL None

PERMEABILIZATION STEP 0.5% Triton-x 100

BLOCKING CONDITIONS Blocked cells in wells with 1% BSA and 10% FCS in PBS for 1hr before adding primary.

SECONDARY ANTIBODY AB6793 antibody texas red sheep anti-rabbit. Used at either 1/200 or 1/1000 diluting with blocker left for 2hours at room temp. Washed with PBS for 45 min.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? First I incubated with primary for 1 hour then I changed to overnight. Before I didn't permeabilise but I changed this the second time.

ADDITIONAL NOTES Permeabilised cells at secondary of 1/200 looked better than at 1/10000 that weren't permeabilised but Texas red was blurry and not too bright. Unfortunately, secondary alone controls also had non-specific staining. This staining looked like cells which were also treated with the primary antibody. The cells I am looking at are mouse somatotropes from the pituitary gland and these should have somatostatin receptor on the surface. Therefore there should be staining. These cells are also conjugated with GFP.

ANSWER:

I'm sorry to hear you are experiencing problems with ab2366 in immunocytochemistry.

This antibody has not been tested in this application, therefore I am unable to provide a specific protocol for this staining method, but I after consultation with the lab that tested the antibody I hope that the following protocol modifications suggestions will help improve your signal:

-the main recommendation would be to try a different secondary antibody since a no primary control gave non specific binding

-fix your cells with ice cold acetone or methanol for 5-10mins. The problem may be that the PFA is over-fixing the protein so the antibody cannot recognize it (and the secondary binds too). It may be worth trying different fixation times with the PFA and the acetone too.

-incubate the primary antibody and secondary antibody in PBS only (no blocking agent) as this can cause background issues.

-try blocking in 5% serum for 1 hour.

-wash in frequent short steps to remove the excess antibody.

Finally it would be worth checking that your mouse cells express a high-enough level of somatostatin receptor as if the expression level is low the antibody may not be able to detect the protein. Mouse pancreatic cells would be the most suitable positive control.

Please do not hesitate to contact me if you have any questions or need further assistance and I'll do my best to help you.

Customer Question: Could you please tell me if this product is the one produced according to S.Schulz?

ANSWER:

Thank you for your enquiry. This antibody was produced in-house. The paper by Schulz listed on the data sheet, is only a general reference concerning the receptor. If you require further information, please contact us again and we will gladly assist you.

Is this antibody (ab2366) directed against all somatostatin receptors, or only one subtype?

Thanks!

ANSWER:

This antibody is directed against somatostatin receptor 1.