Immunofluorescence
Antibodies were tested on XTC (Xenopus laevis fibroblast) cells fixed with methanol or PFA. If the antibody doesn’t work with a fixation condition in a mammalian cell line, the condition was not tested in XTC cells.
| Structure | Antibody | Immunofluorescence | |||||
| Antigen | Host | Source | MeOH | PFA/ TX100 |
Other PFA | Dilution |
|
| Microtubules | Acetyl TUBA4b (Acetylated alpha tubulin) | rabbit mAb | Cell Signalling (Lys40) (D20G3) | Yes | Yes (Brighter than MeOH) | 1/200 | |
| TUBA4b (alpha tubulin) | mouse mAb | Keith Gull. Hybridoma cell line available from Sigma: 00020911 | Yes | Yes | 1/200-1/500 | ||
| Centrosomes |
CEP192 | rabbit pAb | Bethyl A302-324A | No | Not tested | 1/500 | |
| PCNT (Pericentrin) | rabbit pAb | Abcam ab4448 | No | No | |||
| +Tips | MAPRE1 (EB1) | mouse mAb | Cell Signalling IAII/4 | Yes (lovely!) | Not Tested | 1/500 | |
| MAPRE1(EB1) | rat mAb | KT51 Invitrogen/Thermo MA1-72531 | Yes | Not tested | 1/200 | ||
| Spindle etc. | NUMA | mouse mAb | Oncogene Ab-1, #NA08, clone 204-41 | No (but works in eggs & embryos!) | No | 1/100 | |
| EG5/KIF11 | KIF11 | rabbit pAb | Sigma HPA010568 | Not clear | Not clear | tried 1/200 & 1/500 | |
| KIFC1/HSET | KIFC1 | mouse mAb | clone2B9. Sigma, WH0003833M1 or VWR/Avantor USBI128825 | No | No | 1/500 | |
| Kinesin-1 | KIF5B | rabbit mAb | Cell Signaling. E4A5A. Region around Gly408 | No | No | 1/200 | |
| Map7 |
MAP7 | rabbit mAb | Cell Signaling 88646 | No | No | 1/200 | |
| MAP7 (E8K70) | rabbit mAb | Cell Signaling 53626 | No | No | 1/100 | ||
| Dynein |
DYNC1LI1 (LIC1) |
rabbit pAb | Sigma Prestige HPA035013 | Inconclusive | Not tested | 1/200 | |
| DYNC1LI1 (LIC1) | rabbit pAb | Cambridge Bioscience HPA035013 | No | Not tested | 1/200 | ||
| DYNC1LI1 (LIC1) | rabbit mAb | Abcam ab157468 (EPR11240) | Positive in cytosol, faint spindles | Yes but high background, faint spindles | 1/400 | ||
| DYNC1LI1 (LIC1) | rabbit pAb | Thermo Fisher PA-31644 | No | Not tested | 1/200 | ||
| Actin | ACTB AC-74 (Beta actin) | mouse mAb | Sigma A5316 | No | No | 1/250 | |
| Cytokeratin | pan-cytokeratin | mouse mAb | Sigma C2562 | No | Not tested | 1/100 | |
| Vimentin | 13.2 | mouse mAb | Sigma V5255 | No | Not tested | 1/200 | |
| Adherens junctions | CTNNB1 (Beta Catenin) | rabbit mAb | Epitomics 12447-1 | Yes | Yes but also stains nucleolus | 1/100 | |
| Plasma membrane |
CAV1 (Caveolin1) | rabbit pAb | Cell Signaling 3238 | No | No | 1/200 | |
| GJA1 (Connexin 43) | rabbit pAb | Cell Signaling 3512 | No | No | 1/75 | ||
| Nucleus and nuclear envelope |
RANBP2 | rabbit | Bethyl A301-796A | Weak NE in some cells | Weak, possibly +’ve | 1/200 | |
| EMD (Emerin) | rabbit pAb | Abcam ab40688 | No | No | 1/200 | ||
| XPO1 (CRM1) | rabbit pAb | Bethyl A300-469A | Nuclear staining but not NE | Nuclear staining but not NE | 1/500 | ||
| Golgi |
GOLGA2 (GM130) | mouse mAb | BD Biosciences 610823 | Yes | Yes (beautiful!) | 1/100 | |
| AP1 gamma adaptin | mouse | BD Biosciences 610385 | No (very strong mitochondria) | No | 1/500 | ||
| GOLGB1 (Giantin) | rabbit pAb | Abcam ab93281 | No | No | 1/250 | ||
| Early endosomes |
EEA1 | rabbit mAb | Cell Signaling C45B10, 3288S | Not tested | No | 1/200 | |
| EEA1 | mouse mAb | BD Biosciences 610457 | Not tested | No | 1/250 | ||
| RAB5A (RAB5) | mouse | BD Biosciences 610725 | Not tested | Not tested | No (SDS/TX100 permeabilisation) | 1/150 | |
| RAB5A (RAB5) | rabbit mAb | Cell Signaling #3547 | Not tested | Not tested | Yes (SDS/TX100 permeabilisation) | 1/150 | |
| Recycling endosomes | TFR | mouse mAb | Zymed 13-6890 (clone H68.4) | No | No | 1/200 | |
| Late endosomes/ lysosomes | CD63 | mouse mAb | Millipore CBL553 (clone RFAC4) | No | Not tested | 1/200 | |
| Lysosomes |
LAMP1 | mouse mAb | DSHB H4A3 | No | Not tested | 1/1000 | |
| LAMP1 | rabbit | Cell Signaling D2D11 | No | Not tested | 1/500 | ||
| Mitochondria |
HSP70 | mouse mAb | Aff Bio MA3-028 | Yes (beautiful!) | Yes | 1/200 | |
| cytochrome c | mouse | BD Biosciences 556432 | No | No | 1/500 | ||
| COX4I1 (3E11) | rabbit mAb | Cell Signaling 4850 (works in human to zebrafish) | Weak | No | 1/150 | ||
Western Blotting
| Antigen | host | source | works? | notes | dilution |
| HSPA5 (GRP78/BIP) | mouse | Proteintech 66574-1-Ig | yes | 1:10000 | |
| RAC1 | mouse | Proteintech 66122-1-Ig | no | 1:5000 | |
| RUNX1T1 | mouse mAb | Proteintech 67086-1-Ig | no | 1:3000 | |
| XPO1 (CRM1) | mouse mAb | Proteintech 66763-1-Ig | no | 1:10000 | |
| GRB2 | mouse mAb | Proteintech 66880-1-Ig | no | 1:5000 | |
| GOSR2 | mouse mAb | Proteintech 66134-1-Ig | no | 1:3000 | |
| PDIA2 (PDI) | mouse mAb | Proteintech 66422-1-Ig | yes | 1:10000 | |
| CANX | mouse mAb | Proteintech 66903-1-Ig | yes | 1:5000 | |
| mTOR | mouse mAb | Proteintech 66888-1-Ig | no | 1:5000 | |
| RAB27B | mouse mAb | Proteintech 66944-1-Ig | no | 1:1000 | |
| RAB27A | mouse mAb | Proteintech 66058-1-Ig | no | 1:20000 | |
| RAB5A | mouse mAb | Proteintech 66339-1-Ig | no | 1:4000 | |
| DCTN2 (Dynamitin) | mouse mAb | Proteintech 60257-1-Ig | yes | 1:500 | |
| FLII | mouse mAb | Proteintech 67039-1-Ig | yes | 1:10000 | |
| VEGFC | mouse mAb | Proteintech 67116-1-Ig | no | 1:3000 | |
| GORASP1 | mouse mAb | Proteintech 66651-1-Ig | no | 1:4000 | |
| SUMO2/3 | mouse mAb | Proteintech 67154-1-Ig | no | 1:3000 | |
| SMAD1 | mouse mAb | Proteintech 66559-1-Ig | no | 1:5000 | |
| SMAD5 | mouse mAb | Proteintech 67052-1-Ig | no | 1:3000 | |
| STX6 | mouse mAb | Proteintech 60059-1-Ig | no | 1:2000 | |
| RAB18 | mouse mAb | Proteintech 60057-1-Ig | no | ||
| ORAI1 | mouse mAb | Proteintech 66223-1-Ig | no | 1:20000 | |
| STIM1 | mouse mAb | Proteintech 66189-1-Ig | no | 1:500 | |
| FAK | mouse mAb | Proteintech 66258-1-Ig | no | 1:6000 | |
| SEPT7 | mouse mAb | Proteintech 66542-1-Ig | no | 1:10000 | |
| PALLD | mouse mAb | Proteintech 66601-1-Ig | no | 1:2000 | |
| ITGB3 | mouse mAb | Proteintech 66952-1-Ig | no | 1:3000 | |
| KIF5A | mouse mAb | Proteintech 67009-1-Ig | no | 1:3000 | |
| RANGAP1 | mouse mAb | Proteintech 67146-1-Ig | no | 1:10000 | |
| KCNJ13 (Kir7.1) | rabbit pAb | Alomone Labs APC-125 | yes | 1:200 | |
| GATA4 | mouse mAb | DHSB PCRP-GATA4-1A7 | no | ||
| FOXP3 | mouse mAb | DHSB PCRP-FOXP3-1B2 | no | ||
| RUNX1 | mouse mAb | DSHB PCRP-RUNX1-3D7 | no | ||
| CBFB | mouse mAb | DSHB PCRP-CBFB-1C5 | no | ||
| MEF2C | mouse mAb | DSHB PCRP-MEF2C-1D4 | no | ||
| HOXB9 | mouse mAb | DSHB PCRP-HOXB9-1E6 | no | ||
| HHEX | mouse mAb | DSHB PCRP-HHEX-2B6 | no | ||
| FLI1 | mouse mAb | DSHB PCRP-FLI1-4H6 | no | ||
| IRX5 | mouse mAb | DSHB PCRP-IRX5-1B4 | no | ||
| MYOG | mouse mAb | DSHB PCRP-MYOG-IC5 | no | ||
| HOXB2 | mouse mAb | DSHB PCRP-HOXB2-1F2 | no | ||
| NEUROG3 | mouse mAb | DSHB PCRP-NEUROG3-2A6 | no | ||
| MYOD1 | mouse mAb | DSHB PCRP-MYOD1-2A5 | no | ||
| HAND1 | mouse mAb | DSHB PCRP-HAND1-2A9 | no |
Maller Antibodies
| Antibody | Host | Bleed 1 tested | Works? | Bleed 2 tested | Works? | Notes |
| CHK1 | Rabbit pAb | UCO33 Bl.9 | No | UCO33 Bl.4 | Yes | |
| MAPK1 | Rabbit pAb | R1485 Bl.4 | No | R1486 Bl.3 | Yes | background, strong band at 100kDa |
| WEE1 | Rabbit pAb | R7555 BL.3 | Yes | many background bands | ||
| TPX2 | Rabbit pAb | UCO81 Bl.7 | No | UCO80 Bl.3 | No | |
| CCNA2 (cyclinA2) | Goat pAb | G29 Bl.7 | No | G29 Bl.5 | No | |
| CCNB1(cyclinB1) | Sheep pAb | 540 Bl. 13 | Yes | |||
| CCNE1 (cyclinE1) | Rabbit pAb | R3207 Bl.4 | No | R3210 Bl.2 | No | |
| CCND2 (CYCLIN D2) | Rabbit pAb | R7146 Bl.3 | No | |||
| PLK4 (PLX4) | Rabbit pAb | UCO176 Bl. 7 | No | UCO175 Bl.6 | Yes | Some background but strong band just over 100 kDa |
| FBXO5 (EMI1) | Rabbit pAb | UCO55 Bl. 7 | No | UCO55 Bl.5 | Yes | Some background but strong band between 35 and 55 kDa |
| STK10 (Plkk1) | Rabbit pAb | R6958A Bl.7 | No | R6929A Bl.8 | No | |
| CDCA2 (repoman-c) | Rabbit pAb | GN21/822 Bl.3 | No | GN21/821 Bl.2 | No | |
| CDCA2 (repoman) | Rabbit pAb | UCO156 Bl.5 | No | |||
| MCM6 | Rabbit pAb | R6992 Bl.3 | Yes | |||
| CDC25C | Rabbit pAb | R | No | R18376 Bl.3 | Yes | |
| CDC25A | Rabbit pAb | R5860 Bl.7 | No | R5861 Bl.3 | Yes | |
| CDC25 N-term | Rabbit pAb | R849 Bl. 3 | No | |||
| CDC25 C-term | Rabbit pAb | R18376 Bl. 3 | No | |||
| CDC27 | Rabbit pAb | R7514 Bl.7 | No | R7512 Bl. 4 | Yes | |
| CDK1 (CDC2) | Rabbit pAb | R500 Bl.3 | Maybe | double band of right size but many additional bands | ||
| CDC7 | Rabbit pAb | R6710 Bl.4 | No | |||
| CDKNX (Xic1, p27) | Rabbit pAb | R5535 Bl.14 | No | R5655 Bl. 3 | No | |
| CDKNX (Xic1, p27) | Rabbit pAb | R5835 | No | Single strong band, but around 40kDa (should be 27 kDa) | ||
| STK11 (p49, XEEK1, LKB1) | Rabbit pAb | R3955 Bl.6 | No | |||
| MPR-nt | Rabbit pAb | UCO109 Bl.3 | No | |||
| MOS (C-MOS,p39) | Rabbit pAb | R517 Bl. 6 | Yes | |||
| MYT1 | Rabbit pAb | UCO39 Bl.5 | No | |||
| MXD1 (MAD1) | Rabbit pAb | UCO34 Bl.4 | No | |||
| RPS6KA1 (p90-RSK)) | Rabbit pAb | R2188 Bl.3 | Yes | |||
| CCNA1 (CYCLIN A1) | Sheep pAb | 506 Bl.22 | No | 510 Bl.22 | No | |
| CCNB2 (CYCLIN B2) | Sheep pAb | 520 Bl. 3 | Yes | |||
| RPS27 (MPS1) | Rabbit pAb | UCO93 Bl.3 | No | |||
| BCL2 | Rabbit pAb | R5857 Bl.3 | No | |||
| PPL | Rabbit pAb | R1238 Bl.3 | No | |||
| MPRB2 | Rabbit pAb | Uco58 Bl.3 | No | |||
| MPRB3 | Rabbit pAb | UCO56 Bl.3 | Yes | |||
| CDK4 | Rabbit pAb | R5117 Bl.3 | Yes | |||
| MAP2K1 (MEK1) | Rabbit pAb | R1221 Bl.2 | Yes | |||
| BUB1 | Rabbit pAb | R6994 Bl.3 | Maybe | Faint band of other size but many other smaller bands | ||
| BUB3 | Rabbit pAb | R7405 Bl.7 | No | R7407 Bl.3 | Yes | |
| MASTL (GREATWALL KINASE) | Rabbit pAb | UCO162 Bl.3 | Yes |
