HeLa cells transiently transfected with MannosidaseII-tdTomato (Golgi). Primary antibody: [5F8] 1:1,000 Secondary antibody: goat anti-rat Alexa Fluor® 647 Fixation: 3.7 % PFA Scale bar: 10 µm

Dilution series of EGFP in HEK 293T cell lysate Primary antibody: [3H9] 1:1,000 Secondary antibody: goat anti-rat Alexa Fluor® 488 1:1,000

Dilution series of mCherry in HEK 293T cell lysate Primary antibody: [6G6] 1:1,000 Secondary antibody: goat anti-mouse Alexa Fluor® 647 1:1,000

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

Immunofluorescence Primary antibody: Anti-GFP PABG1 1:1,000 Secondary antibody: anti-rabbit_Alexa488 1:500

Western Blot Primary antibody: Anti-GFP PABG1 1:1,000 Secondary antibody: anti-rabbit_Alexa647 1:2,000

Western Blot Primary antibody: Anti-GFP PABG1 1:1,000 Secondary antibody: anti-rabbit_Alexa647 1:2,000

Western Blot analysis of cell extract from HEK293Tcells expressing PCNA-myc

Immunofluorescence of HeLa cells transiently expressing MAG-myc

Western blot analysis of cell extract from HEK293T cells expressing GFP-HA (29.9 kDa)

Immunofluorescence of HeLa cells transiently expressing HA-TOMM70

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 analysis of cell extract from HEK293T cells transiently expressing Actin-Chromobody-EGFP-Halo-tag and from untransfected cells.

Sensitivity tested with purified recombinant Halo-tag protein. Primary antibody: anti-Halo 1:500. Secondary antibody: anti-mouse Alexa Fluor? 647 1:1,000.

Western blot analysis of cell extract from BL21 cells expressing GST

Western blot, dilution series of purified recombinant GST-protein, anti-GST antibody

Western blot analysis of cell extract from HEK293T cells transiently expressing SNAP-Tubulin and from untransfected cells.

Sensitivity tested with purified recombinant SNAP-tag protein

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.