Date post: | 19-Dec-2015 |
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What is green fluorescent protein (GFP) ?
• GFP is a 238-amino acid protein originally isolated from the bioluminescent jellyfish Aequoria victoria
• Can be expressed in any organism without additional cofactors
• Structurally, GFP represents a new motif which has been named the “β-can”
• The “can” consists of 11 β-strands and a central α-helix
Structure of GFP
• The fluorophore is derived from the tripeptide -Ser65-Tyr66-Gly67-
• Formation of the fluorophore occurs in an autocatalytic manner
Structure of the GFP-fluorophore
Antibodies used for fluorescenceList of recommended antibodies used for immunfluorescence
E-GFP Alexa 488 Cy3 Rh-Red-X Alexa 568 Rhodamin Tx-Red Cy5 / Alexa 647Absorbtion 489 499 525/553 540/571 545/577 558 556/589 612/653Emisssion 508 519 575 591 603 574 615 669
anti-Maus AK190 AK183 AKO57 AK202
anti-Rat AK184 AK198 AK154 AK169
anti-Rabbit AK187 AK043 or AK053 AKOO2 AK048 or AK049 AK182
The choice of the flourochrome:
triple-labelling
Alexa 488 for green signalRh-Red-x or Alexa 568 for red signalCy5 for infrared
Cy3 will be the best fluorochrome for the red channel but it will also show up weakly in the green channelTx-Red is not good for the new set of filtersRhomdamin might be o.k.