Does PMKT-140AP contain 0.02% sodium azide?
The product comes in liquid form and is stabilized for long-term storage. The product cannot be lyophilized to powder form because it contains glycerol as stabilizer. We can provide you this antibody in PBS without glycerol for lyophilization as a special case however, the minimum quantity for such a batch will be 2mg.
I run cell lysates from different human cell lines corresponding to 50 ug total protein (lysis buffer 25 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100 with protease and phosphatase inhibitors) under reducing conditions on a 8% gel followed by blotting to a PVDF membrane. As blocking reagent I used 5% skim Milk in 50 mM Tris pH 8, 150 mM NaCl, 0.5% Tween-20, 1 h RT. Followed by 3 washes with TBST buffer (50 mM Tris pH 8, 150 mM NaCl, 0.1% Tween-20).I incubated the blot with the above mentioned antibody overnight at 4 C on a rotating shaker diluted 1:750 in BSA buffer (5% BSA in 50 mM Tris pH 8, 150 mM NaCl, 0.5% Tween-20 ). Next day, followed by 6 washes with 50 mM Tris pH 8, 150 mM NaCl, 0.5% Tween-20. As a secondary antibody I used an anti-rabbit HRP conjugate from cell signaling, diluted 1:10000 in the blocking buffer. Incubation time again 1 h RT. The membrane was washed 6 times as above and developed using ECL. After phosphoMer detection, the blot was washed 6 times with 50 mM Tris pH 8, 150 mM NaCl, 0.5% Tween-20 was analysed for total MERTK using the AF891 from R & D system. Similar process was used for total MERTK also.
Results- When detecting phospho Mer I see several bands from 130 KD to 250 KD. whereas in total MERTK I see a single band.
We now know for sure that:
phospho-MER protein showed some abnormality in the migration on SDS-PAGE, so therefore the proteins will not be exactly at the same place in phospho-lane. The non-phospho-MER has differences in the MW due to differential glycosylation on the core protein. This was very elegantly published by Doug Graham (who cloned this gene) using some of our reagents
I am not sure about the characteristics of non-phospho-MER antibodies used in these experiments. Do they recognize all forms of differentially modified MER or just recognize the core proteins and do not recognize the fully mature MER proteins in the samples? Our non-Phospho-MER antibodies (MKT-101AP, MKT-112AP and MKT-121AP) recognize not only the core proteins but also the glycosylated from different tissues and cell extracts.
In some samples our Phospho-mer antibody (PMKT-140AP) also recognizes a smaller (just above 90kDa) band. The MW of these proteins are apparent in relation to the MW standards and cannot be considered as actual size of the proteins. These numbers will vary quite significantly on different pore size gels, and sometimes even when different companies MW markers are used. We also know that calculated MW will also vary when colored and non-color MW makers are compared on the same gel.
Phospho-MERTK Antibody FITC
Phospho-MERTK Antibody BIOTIN
Phospho-MERTK Blocking Peptide
Phospho-MERTK Positive Control
