Immunochemical detection of peptides and proteins on press-blots after direct tissue gel isoelectric focusing.
A sensitive method is described for the detection of tissue peptides and proteins. They are separated by tissue isoelectric focusing using thin large-pore polyacrylamide gels, containing detergent and dimethylformamide, and are fixed with either glutaraldehyde or formaldehyde in gelatin-coated nitrocellulose membranes using press-blotting. The fixed peptide and protein antigens are visualized by immunoperoxidase staining. The spectrum of fixed tissue constituents may also be used to test antiserum reactivity and specificity in immunocytochemical staining procedures. Isoelectric focusing of 2 microL homogenates of the neurointermediate lobe of the pituitary allowed the immunodetection of peptides and proteins of various sizes and the determination of isoelectric points. However, direct application onto gels of small pieces of frozen tissue sections, sliced in a cryostat, appeared to be more efficient. By direct tissue isoelectric focusing of brain tissue, peptides were effectively eluted and separated from sections up to 100 microns thickness. This allowed the detection of small peptides with a detection limit of approximately 10 pg/section.
van der Sluis
P J
PJ
Netherlands Institute for Brain Research, Amsterdam.
Pool
C W
CW
Sluiter
A A
AA
D000818:Animals; D003101:Collodion; D005557:Formaldehyde; D015151:Immunoblotting / Q000379:methods*; D007120:Immunochemistry; D007525:Isoelectric Focusing / Q000379:methods*; D008297:Male; D010455:Peptides / Q000302:isolation & purification*; D010902:Pituitary Gland / Q000032:analysis; D011506:Proteins / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains
D010455:Peptides; D011506:Proteins; D005557:Formaldehyde; D003101:Collodion