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I am using the newly purchased anti-calcineurin A antibody (ab71149), according to the data sheet, I should see a band at 61 kDa (observed size & 59 kDa being the predicted size). But I see two strong bands at around 60 kDa and another band at 50 kDa. Which one is the calcineurin A specific band? Have you ever observed this? Please do help me with more detail. Thank you in advance.
Asked on Sep 29 2011
Thank you for your inquiry. We have not seen a banding pattern similar to what you're describing with in-house testing. We have predominantly seen a single band at 60 kDa. However, we have heard from a customer who saw something similar to what you're describing using fibroblast cell lysate. https://www.abcam.com/index.html?pageconfig=reviews&intAbID=71149&strFilterApplication=Western blot Important to note is that this protein does have two isoforms, at 57 and 59 kDa, respectively. There are a few things that may be going on here, including degradation of 1 isoform, resolution of the isoforms in an unexpected way due to experimental variances in gel percentage, running buffers, etc. Therefore, I would be inclined to think that both bands are specific to calcineurin A. You may want to run a positive control with HeLa, 293T, or NIH 3T3 cells. Also, take a look at this reference which used this antibody in WB to make sure your protocol is similar: http://www.ncbi.nlm.nih.gov/pubmed/21323745?dopt=Abstract Below is the protocol that was tested in-house: Reagents Needed: 20X Running Buffer Tricine (free base) 71.7 g Tris (free base) 72.6 g SDS 10.0 g Sodium Bisulfite 2.5 g Adjust to 500 ml with ultra pure water. Store at 4°C. For 1X Running Buffer, add 10 ml of 20X Running Buffer to 190 ml of distilled water. 10X Transfer Buffer Tris (free base) 15.2 g Glycine 72.1 g SDS 5.0 g Ultra pure water to 500 ml 10X Transfer Buffer Store at 4°C. 1X Transfer Buffer 10X Transfer Buffer 50 ml Methanol 100 ml Distilled water 350 ml Make fresh for each use. 5% non-fat dry milk in TBST Carnation non-fat dry milk 50 g TBST 1 liter TBST (Tris Buffered Saline with Tween 20, pH8.0) Tris 6.1 g NaC l 8.68 g Tween-20 500 mcl Adjust the volume to 1 liter with distilled water. Adjust pH to 8.0 with HCL. Store at 4-25°C. Electrophoresis in SDS-Polyacrylamide Gel and Transfer to Nitrocellulose: Polyacrylamide gels (4-12% Tricine), running buffer and transfer buffer SeeBlue2 and HiMark molecular weight markers Nitrocellulose membranes Cut open the package that contains the gel cassette and drain away the buffer. Rinse the wells with distilled water. Rinse the wells with fresh 1x running buffer. Place the gels on the buffer core so that the shorter plates face inward. If only using one gel, use a buffer dam to seal the other side. Fill the inner core with fresh 1X running buffer. If there are no leaks, fill the outer core with running buffer. Load samples. Run the gels at 150V until the dye front reaches the bottom of the gel (approximately 60 minutes). Soak nitrocellulose membrane and blotting paper in 1X transfer buffer for at least 5 minutes prior to opening gel cassette. Open gel cassettes and place the gel on the nitrocellulose membrane sandwiched between two pieces of blotting paper. Place in transfer apparatus and fill with fresh 1X transfer buffer. Run transfer apparatus for 60-75 minutes on 35V. Western Blotting: Remove the membrane from the transfer apparatus and place in 20 ml of 5% non-fat dry milk in TBST for one hour, with gentle shaking. Dilute the primary antibody in 15 ml of 5% non-fat dry milk in TBST. For best results, the optimal dilution of antibody should be empirically defined. Incubate the membrane in diluted primary antibody for two hours to overnight with gentle shaking at room temperature. Wash the membrane three times, 10 minutes each time in TBST. Dilute the secondary HRP conjugated antibody in 15 ml of reconstituted 5% non-fat dry milk in TBST. For best results, the optimal concentration of the secondary HRP conjugated antibody should be empirically defined. Incubate the membrane in diluted anti-Rabbit IgG-HRP Conjugate for 60 minutes. Wash as directed in step 4. Develop blots with substrate solution and place in plastic membrane protector. Expose membrane to film or CCD camera. I hope this information helps. If you're still having difficulty, please let me know and I will be happy to help you further.
Answered on Sep 30 2011