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1、TECHNICAL SERVICE GUIDE: WESTERN BLOTTINGCatalog #: sc-53564 Lot #: f0314A. Problem and Previous Experience1. What is the specific problem you are experiencing?The corresponding protein is not detected with the mouse anti-caveolin-1 purchased from your company, but for the same piece of film, corres
2、ponding protein has been detected with the original saved rabbit anti-caveolin-1 in our laboratory. In addition, the secondary antibody, the goat anti-mouse antibody, which was used to detect caveolin-1 had successfully detected the proteins such as GAPDH, -acting, SMA and so on. (proved the seconda
3、ry antibody have no problem).2. What size bands were expected and what size bands were detected?21KD protein is what we expected, actually, we hadnt detected any stripe in any size.3.Was the blot blank or was a dark background or non-specific bands seen?The blot is blank.4. Did this same vial of pro
4、duct work in the past? What were the results?We had never seen any blot detected, using your antibody.5. Did other lots of this product work in the past? Which lots? What were the results? Using the anti- rabbit caveolin-1 produced by other companies, corresponding protein was detected.B. Samples an
5、d ControlsFrom what species (animal) was the sample and what type of sample was used (cell lysate, tissue, purified protein, etc.)? What lysis buffer was used?Rat and human origin cell lysates were used to detect. We used the lysate of RIPA.1. How much sample was loaded on the gel? 3. If the blockin
6、g peptide was used, was it used as a negative control?None4. What positive and/or negative controls were used, and what were the results?For the same piece of film, corresponding protein had been detected with the original saved rabbit anti-caveolin-1 in our laboratory. In addition, the secondary an
7、tibody, the goat anti-mouse antibody, which was used to detect caveolin-1 had successfully detected the proteins such as GAPDH, which had proved that the secondary antibody had no problem.C. Blocking1. What were the blocking conditions (blocking solution, how long, what temperature. etc.)?5% skim mi
8、lk-TBS, 1 hour, at room temperatureD. Primary and Secondary Antibodies1. At what dilution(s) was the primary antibody used and what was the diluent?TBS dilution2. What were the incubation conditions (time, temperature) for the primary antibody?4 overnight3. From which company was the secondary antib
9、ody obtained and what type of secondary was being used (HRP or AP conjugated, anti-rabbit or anti-goat, etc.)? What detection system was used, and was it compatible with the secondary conjugate?Secondary antibody was HRP anti-mouse, purchased from biological UNOCI. the detection system had detected
10、the marker of the secondary.4. At what dilution(s) was the secondary antibody used and what was the diluent?TBS dilution5. What were the incubation conditions (time, temperature) for the secondary antibody?Incubated for 1.5 hours at 376. Has a secondary control been performed successfully?Other appr
11、opriate proteins can be successfully detected by the secondary antibody. 7. If using Cruz Markers, how many mL were loaded, and was a Cruz Marker compatible secondary antibody used?The band had not been detected in any 1 : 1000, 1: 500,or 1: 200 fold dilution. .8. Can you verify that all Molecular W
12、eight Markers transferred to the membrane?Definite9. At what temperature has the primary antibody been stored? Has it ever been frozen?Store at 4.E. Membrane, Gel, Detection1. What detection system was used and what were the exposure times for the film?Tanon 6200 Chemiluminescent gel imaging system,
13、 not any blot had been seen with exposure time in 10,30,60,100,300s.2. What type of membrane was used (PVDF or nitrocellulose)?PVDF 3. What percentage gel was used? Was this appropriate for the size of the protein?10%, appropriate.F. Preferred replacement: （请客户列出自己想要的优先赔付方式）In order of preference, f
14、rom high to low, please list your desired replacement for this antibody: A, a new lot of the same antibodyB, different antibody for the same protein (please specify catalog #)C, any other antibody from our catalog (please specify catalog #)G. Related photos Please provide with whole images notcut visions.（请客户提供完整的图片）1. Using your caveolin-1 antibodies, we had not seen a bands 2、Using the surviving anti- rabbitcaveolin-1 in laboratory for the same film, the blot was detected3. Using the goat-anti-mouse secondary antibody being used in figure II, the blot had been seen.