Anti-Calnexin抗体(ab13505)

Thank you for your email and patience, and I'm sorry to hear that you are experiencing difficulty with this antibody. Below I have included the WB protocol that the originator used when testing ab13505. Please note that this is a general protocol, and a specific WB protocol is not necesary for this antibody. The antibody was characterized in WB using Heat Shocked HeLa Cell Lysate and this is recommended as a positive control. I would also suggest incubating with the primary antibody for overnight at 4C at a dilution of 1:1000. Please also ensure that you are loading suffucient amount of protein (20-30 ug lysate) and that the protein transferred properly to the membrane. Please also check that your secondary antibody is working. I hope that this helps, and please contact us again if you have any additional questions. Western Blot Protocol The immunobloting technique described is separated into five steps: gel electrophoresis, protein transfer, blocking, addition of antibody and detection. It is advisable to run internal standards when possible to monitor each step of the process. A source of antigen known to react with the antibody should always be included as a positive control for the antibody being used. Gel Electrophoresis – Reducing conditions 1.Determine protein concentration using an assay such as Lowry or Bradford. 2.Boil protein samples in the smallest possible volume of Laemmli sample buffer for 5 minutes immediately after harvesting (Laemmli, 1970). Some antigens are heat labile and heating at 70?C is recommended. 3.Load protein samples and controls. Determine appropriate protein by running preliminary gels and staining with Coomassie Blue. Include unstained or pre-stained molecular weight markers. Pre-stained molecular weight markers will verify that the gel has run properly. 4.Start electrophoresis at 100V. After the dye front has moved into the separating gel, increase to 200V. Stop electrophoresis when the dye front reaches the bottom of the gel. Protein Transfer 1.Transfer separated protein bands onto a suitable membrane, such as nitrocellulose paper or PVDF (for small MW proteins). 2.In semi-dry transfer , the gel and membrane are equilibrated in transfer buffer for 30 minutes prior to transfer. Semi-dry transfer is run for 30 minutes at 20V at room temperature. 3.Equilibration of the gel and membrane is not necessary when using a submerged transfer. With a submerged-type apparatus transfer at 4C for 1 hour at 100V. For submerged transfers, use 1X Towbin Buffer: 1X Towbin Buffer = 25mM Tris + 192mM Glycine + 20% methanol 4.Assemble the apparatus according to manufacturers’ instructions. 5.With either method, at no time should the apparatus or buffer be allowed to become hot. Use a cooling coil or run the transfer in a cold room to avoid the generation of gas bubbles in the sandwich. 6.Verify protein transfer by staining the membrane with Ponceau S stain (0.1% Ponceau S in 5% acetic acid Sigma P 7170). Incubate for ~30 seconds. Mark the MW standards lane with a pencil if pre-stained markers were not used. Destain immediately in PBS/0.05% Tween 20 using agitation. The staining is reversible, and may be repeated if necessary. Blocking 1.Block non-specific binding sites on membrane. Our preferred blocking solution is 5% Blotto: 5% wt/vol nonfat dry milk/0.05% Tween 20/0.02% NaN3 in PBS. Note: thimersol is used in place of azide for immunoblotting using ECL method of color development. The membrane is sealed in a bag containing the solution with as few air bubbles as possible and incubated overnight at room temperature on the rotator. All blocking solutions should be stored at 4?C. Addition of Antibody 1.Incubate the membrane with the primary antibody for 1 hour at room temperature at appropriate dilution in 5% Blotto. Use suggested working dilution stated in Technical Specifications sheet as a starting point for your assay. You may empirically determine a more appropriate dilution, which is dependant on the individual experiment and sample source. If you are using a more sensitive detection system, such as ECL, it may be necessary to use a more dilute preparation. 2.Wash with PBS0.05 Tween 20 three times for 5 minutes. Wash at room temperature with moderate agitation. 3.Incubate the membrane with the second antibody conjugated to the appropriate detection substrate (i.e. AP) for 1 hour at room temperature, as in Step 1. Secondary antibodies from Stressgen are generally used at a dilution of 1:1000 in 5% Blotto. 4.Wash three times with PBS/0.05% Tween 20 for 5 minutes per wash. Detection 1.Proceed with desired detection system. For alkaline phosphatase-mediated color reactions, use NBT and BCIP chromogenic substrates (Stressgen cat# SUB-101) in alkaline phosphatase buffer: APB = 100mM Tris-HCl, pH 9.5, 100mM NaCl, 5mM MgCl. 2.Colored, insoluble reaction products should become visible within 30 seconds to several minutes. To stop the color reaction, decant and wash with distilled water. For storage, the membranes should be air-dried while affixed to heavy filter paper backing to prevent wrinkling. Where desired, photography should be done immediately.