Anti-CACNA1G + CACNA1H antibody (ab81099)

ReviewID: xxx AbID: 81099, Anti-CACNA1G + CACNA1H antibody Rating: Inconclusive Image: Anti-CACNA1G + CACNA1H antibody for Immunohistochemistry (Frozen sections) (Mouse) Sample: Species: Mouse Type: Tissue sections Specification: heart Application: Application: Fixative: Paraformaldehyde Permeabilization: Yes Blocking step: BSA as blocking agent for 1 hour at 25°C Other detail: Dilution: 1/250 Incubation time: 12 hours at 4°C Secondary Antibody: Name: Non-Abcam Antibody was used: goat anti rabbit IgG488 Conjugation: Alexa Fluor® 488 Dilution: 1/500 Additional Data: Additional Notes: we tried different concentrations, 1/50-1000 dilution. no specific staining was seen.

ANSWER:

According to our records, ab81099 was proving difficult to use in IHC-Fr and we were in contact in order to help resolve the issue.

Looking at our correspondence, it appears that we are awaiting to hear back in order to help resolve this case.

Since you obtained poor results using the antibody in a tested species and application, we would like to follow up on this to see if we can possibly improve the results you are seeing with this antibody.

Based on the information in your review, I feel that altering the following step in your protocol may help improve your results:

1) Have you tried any other tissue, beside mouse heart? It might be worthwhile to do so.
2) I might suggest trying a no primary control to see if the problem comes from the secondary antibody?
3) Have you tried other fixation reagents, such as acetone or methanol?
4) Was a shorter antibody incubation time tried?

If you decide to follow these suggestions, please let me know if they are helpful. Additionally, this antibody is covered by our Abpromise, so if the suggestions do not help and since the antibody was purchased within the past 6 months, I would be happy to discuss a refund or replacement with you to resolve this issue.

If the requested information has already been sent, it appears that it did not reach our Scientific Support team and we apologize for this inconvenience. In this case we would like to ask for the information again so that we can reach a resolution.

If the issue has already been settled, please let us know so that we can be assured that the problem has been solved to your satisfaction and update our records.

We wish you the best of luck with your research and look forward to a reply.

Immunohistochemistry (Frozen sections) (Mouse) Sample: Species: Mouse Type: Tissue sections Specification: heart Application: Application: Fixative: Paraformaldehyde Permeabilization: Yes Blocking step: BSA as blocking agent for 1 hour at 25°C Other detail: Dilution: 1/250 Incubation time: 12 hours at 4°C Secondary Antibody: Name: Non-Abcam Antibody was used: goat anti rabbit IgG488 Conjugation: Alexa Fluor® 488 Dilution: 1/500 Additional Data: Additional Notes: we tried different concentrations, 1/50-1000 dilution. no specific staining was seen.

ANSWER:

Thank you for submitting an Abreview of ab81099. As you may have noticed, your review has now been published on our website.

Since you obtained poor results using the antibody in a tested species and application, we would like to follow up on this to see if we can possibly improve the results you are seeing with this antibody.

Based on the information in your review, I feel that altering the following step in your protocol may help improve your results:

1) Have you tried any other tissue, beside mouse heart? It might be worthwhile to doso.
2)I might suggest tryinga no primary control to see if the problem comes from the secondary antibody?
3) Have you tried otherfixation reagents, such as acetone or methanol?
4) Was a shorter antibody incubation time tried?

If you decide to follow these suggestions, please let me know if they are helpful. Additionally, this antibody is covered by our Abpromise, so if the suggestions do not help and since the antibody was purchased within the past 6 months, I would be happy to discuss a refund or replacement with you to resolve this issue.

I look forward to your reply.

Thanks for your kindly reply, this customer would likes to know the positive control and negative control of this antibody, could you please offer any selection or suggestion to this customer?   

ANSWER:

T-type channels serve pacemaking functions in both central neurons and cardiac nodal cells and support calcium signaling in secretory cells and vascular smooth muscle. The following link showing the expression level of CACNA1G in different tissues may be useful for your customer : http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Hs.591169

This customer has purchased ab81100 (Anti-CACNA1I antibody), and conducted the WB with rat sample. The results show multiple bands and wrong band size; therefore this customer wants to ask for your help to modify her experiment step, could you please offer any suggestion to improve her wb result? I attached the wb image in this letter and his experiment step as follow:

1. Order details: Batch number: gr34762-3 Po: 961552 Abcam product code: ab81100 Antibody storage conditions (temperature/reconstitution etc) -20oC

2. Please describe the problem (high background, wrong band size, more bands, no band etc). Wrong band size, multiple bands, non-specific, and no major band

3. On what material are you testing the antibody in WB? · Species: rat

· What’s cell line or tissue: Brain cortex, spinal cord

· Cell extract or Nuclear extract:

· Purified protein or Recombinant protein:

3. The lysate How much protein was loaded: 50ug What lysis buffer was used: RIPA buffer What protease inhibitors were used: Roche Protease Inhibitor Cocktail What loading buffer was used: Laemmli sample buffer(5X) Phosphatase inhibitors: Did you heat the samples: temperature and time: 95 oC, 5min

4. Electrophoresis/Gel conditions/ Transfer conditions Reducing or non reducing gel: Reducing Reducing agent: Gel percentage : 7% Transfer conditions: (Type of membrane, Protein transfer verified):PVDF

5. Blocking conditions Buffer: 5% BSA solution, weigh 5 g per 100 ml of Tris Buffer Saline Tween20 (TBST) buffer. Incubation time:1,5hr Incubation temperature:RT

6. Primary Antibody Species:rat Reacts against: rabbit · At what dilution(s) have you tested this antibody: 50mg/ml

· What dilution buffer was used: TBST

· Incubation time: 2hr

· Incubation temperature: RT

· What washing steps were done: 6 times

7. Secondary Antibody (ab6721) Species: goat Reacts against: rabbit At what dilution(s) have you tested this antibody: 1:5000 Incubation time: 1hr Wash steps: 6-10 times Fluorochrome or enzyme conjugate:HRP Do you know whether the problems you are experiencing come from the secondary? no

8. Detection method ECl, ECl+, other detection method: ECl

9. Did you apply positive and negative controls along with the samples? Please specify. GAPDH 10. Optimization attempts · How many times have you tried the Western? 1 time

· Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): no

· Do you obtain the same results every time e.g. are background bands always in the same place? What steps have you altered?

ANSWER:

Thank you for the questionnaire.

Western blotting of big proteins such as CACNA1G and CACNA1H can be difficult but I looked at your blot as well as your protocol again and I think some changes may solve your problems. - I would recommend to incubate the primary antibody overnight at 4°C. - I would suggest to dilute the primary antibody in the blocking solution containing 5% BSA. - I would highly suggest to optimize the transfer conditions by increasing the transfer time to 18 or 24 hours (in some examples, for big proteins, the transfer is done for 48 hours) - A transfer at 30V for a long period of time is fine but it has been shown that a "boost" at 70-80V for 1 hour at the end of the transfer can be of significant improvement. - I am guessing you used SDS in the transfer buffer, which is good. You can use slightly more SDS than normal in the transfer buffer, up to 0.05% and reduce the proportion of methanol to 10%. The reason is that methanol washes out the SDS from the protein.

I hope these suggestions will help to improve the results with this antibody in WB. Please do not hesitate to contact us if you need any more advice or information.