Determination of Antibody Specificity by Western Blotting
Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Research › peer-review
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Determination of Antibody Specificity by Western Blotting. / Celis, Julio E.; Moreira, José M.A.; Gromov, Pavel.
Cell Biology, Four-Volume Set. Vol. 1 Elsevier Science Inc., 2006. p. 527-532.Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Research › peer-review
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TY - CHAP
T1 - Determination of Antibody Specificity by Western Blotting
AU - Celis, Julio E.
AU - Moreira, José M.A.
AU - Gromov, Pavel
PY - 2006/12/1
Y1 - 2006/12/1
N2 - This chapter describes the role of Western Blotting in determining antibody specificity. The procedure is illustrated using whole protein extracts obtained from non-cultured human keratinocytes and the bladder TCC cell line RT4. Proteins are separated by two-dimensional (2D) gel electrophoresis blotted to a nitrocellulose membrane, and developed using the HRP color development reagent and/or the ECL procedure. Place the wet nitrocellulose membrane on top of the gel. The operation should be done under the buffer. Rub the membrane from one end to the other to eliminate bubbles. Primary antibodies should be diluted to a volume of 8 ml. The dilution should be optimized in preliminary experiments for the best results in terms of high signal and low background. Cut the appropriate area of the blot with a scalpel using the X-ray film as reference and wet by capillarity in the skimmed milk solution. Incubate with shaking for 1 h at room temperature or overnight in the cold room in the same solution. © 2006
AB - This chapter describes the role of Western Blotting in determining antibody specificity. The procedure is illustrated using whole protein extracts obtained from non-cultured human keratinocytes and the bladder TCC cell line RT4. Proteins are separated by two-dimensional (2D) gel electrophoresis blotted to a nitrocellulose membrane, and developed using the HRP color development reagent and/or the ECL procedure. Place the wet nitrocellulose membrane on top of the gel. The operation should be done under the buffer. Rub the membrane from one end to the other to eliminate bubbles. Primary antibodies should be diluted to a volume of 8 ml. The dilution should be optimized in preliminary experiments for the best results in terms of high signal and low background. Cut the appropriate area of the blot with a scalpel using the X-ray film as reference and wet by capillarity in the skimmed milk solution. Incubate with shaking for 1 h at room temperature or overnight in the cold room in the same solution. © 2006
UR - http://www.scopus.com/inward/record.url?scp=84884827726&partnerID=8YFLogxK
U2 - 10.1016/B978-012164730-8/50064-2
DO - 10.1016/B978-012164730-8/50064-2
M3 - Book chapter
AN - SCOPUS:84884827726
SN - 9780121647308
VL - 1
SP - 527
EP - 532
BT - Cell Biology, Four-Volume Set
PB - Elsevier Science Inc.
ER -
ID: 221777731