Microsequence analysis of electroblotted proteins. II. Comparison of sequence performance on different types of PVDF membranes.
The influence of different types of polyvinylidene difluoride (PVDF) membranes on gas phase sequence performance has been evaluated. These PVDF membranes have been classified as either high retention (Trans-Blot and ProBlott) or low retention membranes (Immobilon-P) based on their ability to bind proteins during electroblotting from gels. Initial yields, repetitive yields, and extraction efficiency of the anilinothiazolinone amino acid derivatives have been compared for several standard proteins that have been either electroblotted or loaded onto PVDF membranes by direct adsorption. These results show that the major differences in initial sequence yields between membranes arise from differences in the amount of protein actually transferred to the membrane rather than sequencer-related factors. In contrast to several previous observations from other laboratories, more tightly bound proteins do not sequence with lower initial yields and initial yields are not affected by the ratio of surface area to protein. The stronger binding on high retention PVDF membranes does not adversely affect recoveries of difficult to extract, or very hydrophobic, amino acid derivatives. Several amino acids, especially tryptophan, are actually recovered in dramatically higher yield on high retention membranes compared with either Immobilon or glass filters. At the same time, the protein and peptide binding properties of high retention membranes will frequently improve the repetitive yield by minimizing sample extraction during the sequencer cycle. Stronger protein binding together with improved electroblotting yields offer substantially improved sequence performance when high retention PVDF membranes are used.
Reim
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DF
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
Speicher
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DW
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000302:isolation & purification; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006736:Horses; D015151:Immunoblotting / Q000379:methods; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry / Q000302:isolation & purification; D011145:Polyvinyls; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D017421:Sequence Analysis / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000302:isolation & purification; D013844:Thiazoles / Q000302:isolation & purification; D005465:Fluorocarbon Polymers
D001059:Apoproteins; D008567:Membranes, Artificial; D009211:Myoglobin; D011145:Polyvinyls; D011506:Proteins; D012710:Serum Albumin, Bovine; D013844:Thiazoles; C010857:apomyoglobin; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers