The purified complement C3 can be dissociated into four protein bands under reducing and boiled condition. The epitope recognized by GTX02807 C3 antibody [M68] is on the 50-kDa fragment.
The epitope recognized by GTX02807 is significantly destroyed by boiling and reducing treatment and requires much more proteins loaded for the detection.
The protein band 1, 3, 4, 5, and 6 as indicated are sliced out and subjected to MS/MS protein identification, confirming these bands to be complement C3. The relative abundance of peptides matching to C3 is 97% for band 1, 75% for band 3, 98% for band 4, 91% for band 5, and 45% for band 6.
For the best detection sensitivity, the samples should be treated under non-boiled and non-reducing conditions.
Lane A : M68 E2 (contained C3 at highest concentration), 1 μl
Lane B : Boiled sample, 5 μl
Lane C : Boiled and reduced sample, 20 μl

Complement C3 can be detected in human milk by western blot analysis under non-reducing non-boiled conditions using the GTX02807 C3 antibody [M68] as two groups of protein bands.
For the best detection sensitivity, the samples should be treated under non-boiled and non-reducing conditions.
Lane 1 : 10 μl
Lane 2 : 5 μl
Lane 3 : 2 μl

The two groups of complement C3 proteins can be purified by GTX02807 C3 antibody [M68] immunoaffinity chromatography from the partially purified milk fractions. Human milk proteins were loaded onto CM-Sepharose 4B column and proteins, including C3, were eluted by different salt (NaCl) concentration. The slat concentration was smaller than 0.3 N. The CM low salt fractions were collected and loaded to the mAb M68-Sepharose column to purify C3. After washing the immunoaffinity column, the captured proteins were eluted by 0.1 N glycine buffer pH 2.4. The eluates were collected into several fractions, E1, E2, E3, E4, E5 and E6. E1-E4 fractions were analyzed by SDS-PAGE and the eluted proteins were stained by CBB.
The protein band 2 and 7 as indicated are sliced out and subjected to MS/MS protein identification (by Prottech Inc.), confirming these bands to be complement C3. The relative abundance of peptides matching to C3 is 98% for band 2 and 96% for band 7.
For the best detection sensitivity, the samples should be treated under non-boiled and non-reducing conditions.
Lane A : Input (partially purified milk fractions)
Lane B : Flow through
Lane C : Elution 1
Lane D : Elution 2
Lane E : Elution 3
Lane F : Elution 4
Loading : 20 μl