Immunostaining of Drosphila embryos expressing an mNeonGreen tagged protein. Left mNeonGreen, right anti-mNeonGreen antibody 32F6. Corutesy Clara Sidor, MRC-LMB
Immunostaining of HeLa cells transiently expressing mNeonGreen fused to Actin Chromobody (green) with 32F6 anti-mNeonGreen antibody (red). Merge image shows overlay of green and red channels and DAPI (blus). Scale bar, 10 μm
Western blot analysis of N- and C-terminal Spot-tagged GFP added to HEK- 293T cell lysate. Detection with Spot-tag® antibody [28A5] (28a5, ChromoTek) 1:5,000 and Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, Alexa Fluor® 488 [CTK0103, CTK0104] (sms1AF488-1, ChromoTek) 1:5,000. Western blot membrane was incubated simultaneously with the primary antibody and Nano-Secondary® (one-step staining).
Western blot analysis of C-terminal Spot-tagged GFP added to HEK-293T cell lysate. Detection with Spot-tag® antibody [28A5] (28a5, ChromoTek) 1:5,000 and anti-mouse secondary antibody HRP 1:1,000.
Western blot analysis of different cell lysates +/- 50 ng C-terminal Spot-tagged GFP: human (HEK-293T), yeast (S.cerevisiae), insect (High Five™), bacteria (E.coli) and plant (A.thaliana). Detection with Spot-tag® antibody [28A5] (28a5, ChromoTek) 1:5,000 and Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, Alexa Fluor® 488 [CTK0103, CTK0104] (sms1AF488-1, ChromoTek) 1:5,000. Western blot membrane was incubated simultaneously with the primary antibody and Nano-Secondary® (one-step staining).
ELISA detection of N- and C-terminal Spot-tagged GFP with Spot-tag® antibody [28A5] (28a5, ChromoTek). N- and C-terminal Spot-tagged GFP was coated on a microtiter plate, blocked and detected with 1 nM Spot-tag® antibody [28A5] (28a5, ChromoTek) and anti-mouse IgG antibody AP.
Western Blot (WB) analysis of His-eGFP-V5 added to HEK293T cell extract
The anti-V5 antibody [SV5-P-K] is a recombinant, monoclonal mouse IgG1, has very low background and detects even low amounts of V5-tagged proteins. Secondary antibody ChromoTek’s Alpaca anti-mouse IgG1 Alexa Fluor 488 Nano-Secondary.
Western blot detection of fluorescent proteins from different lineages.
SDS-PAGE was performed with 20 ng of purified recombinant proteins per lane. Positive signal could be obtained with red fluorescent proteins from Discosoma dsRed lineage (mCherry, dsRed, tdTomato), with synthetic red fluorescent protein mScarlet, with fluorescent proteins from Entacmaea lineages (mRuby2, mKate2, TagRFP, TagBFP). Purified recombinant green fluorescent proteins EGFP, TurboGFP and mNeonGreen were used as negative controls. Pan-RFP (pabr1) antibody was applied at 1:1000 dilution o/n at +4°C. Secondary antibody: goat anti-rabbit HRP.
Note: Red fluorescent proteins often demonstrate multiple bands on Western blots due to partial fragmentation. These bands correspond to differently truncated forms of red fluorescent proteins.
Immunostaining of HeLa cells, transiently transfected with different fluorescent proteins. Primary antibody: pan-RFP (pabr1) 1:800 for 1 h RT. Secondary: goat anti-rabbit. Upper row shows the innate signals from transfected fluorescent proteins, nuclei are in cyan. Lower row shows the signals from immunostainings with pan-RFP (pabr1) antibody, nuclei are in cyan. Pan-RFP (pabr1) can be used for IF detection of mScarlet, mCherry, tdTomato, mPlum, TagRFP, mKate TagBFP, mRuby2.
Western blot detection of purified mCherry red fluorescent protein down to 1-2 ng. A serial dilution of purified recombinant mCherry protein (250 ng - 1 ng) was subjected to SDS-PAGE. Purified recombinant EGFP (250 ng) was used as negative control (last lane). Pan-RFP (pabr1) antibody was applied at 1:1000 dilution o/n at +4°C. Secondary antibody: goat anti-rabbit HRP.