Figure 3. Fibrinogen anchors facilitate... | Rockefeller University Press

Figure 3.

$Fibrinogen anchors facilitate multiplexed micropatterning of proteins.(A) Scheme illustrating the different steps for multiplexed micropatterning using fibrinogen anchors. Note that the two proteins to be micropatterned (GFP and BSA-biotin-Alexa647) are added together after the micropatterning process. This could help maintain protein activity, as proteins can be added in their optimal buffer (rather than the optimal micropatterning buffer) and are not exposed to UV-induced ROS or the BBTB. (B) Bottom line: Multiplexed micropatterning of GFP and BSA-biotin-Alexa647 (5 µg/ml) with fibrinogen-GBP and fibrinogen-NeutrAvidin anchors (50 µg/ml) using the scheme depicted in A onto PLL-PEG–coated glass. Top and middle lines: Controls of bottom panel by exchanging fibrinogen-GBP with fibrinogen-Alexa546 (top line) or by exchanging fibrinogen-NeutrAvidin with fibrinogen-Alexa546 (middle line) followed by coinjection of GFP and BSA-biotin-Alexa647. Note that there is high specificity of the proteins of interest for their respective anchor and minimum overlap between the two proteins. This implies that the successive micropatterns of fibrinogen anchors have been efficiently quenched (otherwise the proteins of interest could bind to the wrong micropattern). (C) Quantification of the increased specificity of GFP and BSA-biotin-Alexa647 for their respective fibrinogen-GBP or fibrinogen-NeutrAvidin anchor, expressed as the amount protein going to the wrong patterns as a fraction of the amount going to the right pattern (mean ± SEM). Statistics were performed using an unpaired t test. (D) Similarity of the micropatterning homogeneity of GFP on fibrinogen-GBP micropatterns and BSA-biotin-Alexa647 on fibrinogen-NeutrAvidin micropatterns (mean ± SEM). n, number of micropatterns measured. Scale bar, 10 µm.$

Fibrinogen anchors facilitate multiplexed micropatterning of proteins. (A) Scheme illustrating the different steps for multiplexed micropatterning using fibrinogen anchors. Note that the two proteins to be micropatterned (GFP and BSA-biotin-Alexa647) are added together after the micropatterning process. This could help maintain protein activity, as proteins can be added in their optimal buffer (rather than the optimal micropatterning buffer) and are not exposed to UV-induced ROS or the BBTB. (B) Bottom line: Multiplexed micropatterning of GFP and BSA-biotin-Alexa647 (5 µg/ml) with fibrinogen-GBP and fibrinogen-NeutrAvidin anchors (50 µg/ml) using the scheme depicted in A onto PLL-PEG–coated glass. Top and middle lines: Controls of bottom panel by exchanging fibrinogen-GBP with fibrinogen-Alexa546 (top line) or by exchanging fibrinogen-NeutrAvidin with fibrinogen-Alexa546 (middle line) followed by coinjection of GFP and BSA-biotin-Alexa647. Note that there is high specificity of the proteins of interest for their respective anchor and minimum overlap between the two proteins. This implies that the successive micropatterns of fibrinogen anchors have been efficiently quenched (otherwise the proteins of interest could bind to the wrong micropattern). (C) Quantification of the increased specificity of GFP and BSA-biotin-Alexa647 for their respective fibrinogen-GBP or fibrinogen-NeutrAvidin anchor, expressed as the amount protein going to the wrong patterns as a fraction of the amount going to the right pattern (mean ± SEM). Statistics were performed using an unpaired t test. (D) Similarity of the micropatterning homogeneity of GFP on fibrinogen-GBP micropatterns and BSA-biotin-Alexa647 on fibrinogen-NeutrAvidin micropatterns (mean ± SEM). n, number of micropatterns measured. Scale bar, 10 µm.

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