hFAB™ Rhodamine
Housekeeping Antibodies
Catalog #Description

12004163
12004164
12004165
12004166
12004167
12004168

 nti-Actin hFAB Rhodamine Antibody, 200 µl
AAnti-Actin hFAB Rhodamine Antibody, 40 µl
Anti-Tubulin hFAB Rhodamine Antibody, 200 µl
Anti-Tubulin hFAB Rhodamine Antibody, 40 µl
Anti-GAPDH hFAB Rhodamine Antibody, 200 µl
Anti-GAPDH hFAB Rhodamine Antibody, 40 µl

Shelf life: 1 year at –20°C lyophilized; 6 months at 4°C after resuspension.

Instruction Manual
Visit bio-rad.com/web/HKP for more detailed
information about this product.
For technical support call your local Bio-Rad office. In the U.S.
call 1-800-4BIORAD (1-800-424-6723).

Introduction
Bio-Rad’s hFAB™ Rhodamine Antibodies arefluorescently labeled primary antibodies raised against
the housekeeping proteins (HKPs) actin, tubulin, andGAPDH. They are intended to be used for normalizing
protein loading in western blotting experiments.
The hFAB Rhodamine Antibodies are themselves
sufficient and do not need any secondary antibody for
detecting the housekeeping protein. For fluorescencemultiplexing, these hFAB Rhodamine Antibodies
can be incubated along with a variety of fluorescentsecondary antibodies, including our new StarBright™
Blue 700 Secondary Antibodies. hFAB Antibodies
are recombinant antibody fragments that are notrecognized by conventional secondary antibodies,
which means they can be used with primary antibodies
of any species against your target protein.

hFAB Rhodamine Housekeeping Antibodies |2hFAB Antibodies are designed to give a linear
response to HKPs in a range typical for their presencein cellular lysates. They are labeled with a rhodamine
derivative that has minimal spectral cross talk withStarBright Blue 700 Secondary Antibodies. The
fluorophore is excited at around 530 nm (green light)
and emits maximally around 580 nm.
Note: hFAB Housekeeping Antibodies were createdusing human GAPDH, actin, and tubulin sequences
but will recognize their respective targets from othermammalian species. The sensitivity may not be as highfor non-human samples.

hFAB Rhodamine Housekeeping Antibodies |3

Instructions for Use
Preparation
Resuspend the contents of the tube in the indicatedvolume of distilled or deionized water and leave on ice forat least 30 min prior to use. The resuspended solution
may be stored at 4ºC in the dark for up to 6 months. Do
not freeze the resuspended solution.
Brief centrifugation (pulse spin for 2–3 sec at maximum
speed on a tabletop microcentrifuge) may be used tocollect the contents at the bottom of the tube.

General Guidelines
For optimal results, we recommend using low
fluorescence PVDF membranes for transfer.
For blocking and washing, use 2 ml of solution per
10 cm2 of membrane (15 ml for a mini gel). For primaryand secondary antibody incubations, use 1 ml of solution
per 10 cm2 of membrane (10 ml for a mini gel). Use thesmallest flat bottom tray that will accommodate the blot.

hFAB Rhodamine Housekeeping Antibodies |4

Protect the blot from light (for example, using aluminumfoil) during incubations with fluorescent antibodies. Image
immediately after step 5 for best results.
Do not allow the blot to dry out at any time prior toimaging. The blot must be kept moist during imaging
for best results. We recommend using all blue protein
standards since red- or pink-colored standards may
fluoresce brightly in the rhodamine imaging channel andcan interfere with data acquisition.
Shake or rock well (without spilling) during incubations.
hFAB Rhodamine Antibodies may be used successfullywith StarBright Blue 700 Secondary Antibodies (andother fluorescent secondary antibodies, preferably withemissions >650 nm) in many different immunodetection
protocols. The following protocol is recommended fordetection with high sensitivity, low background, andminimal nonspecific cross-reactivity.

hFAB Rhodamine Housekeeping Antibodies |5Protocol
1. Block: 1 hr at room temperature (RT) with Tris
buffered saline (TBS) + 1% casein.
2. Incubate in primary antibody for target protein:
Dilute and incubate the primary antibody as
specified in your protocol or by the vendor. If noprotocol is provided with the primary antibody, it
may be diluted in TBS + 1% casein buffer.
3. Wash: 5 x 5 min at RT with TBST (TBS + 0.05%Tween 20).
4. Incubate with hFAB Rhodamine Antibody:
Dilute both reagents into TBS + 1% casein.
The recommended starting dilution for hFAB
Rhodamine Antibody is 1:1,000 (range 1:1,000–1:10,000). If detecting other target proteins usingthe StarBright Blue 700 Secondary Antibody, therecommended starting dilution is 1:2,500 (range1:2,500–1:5,000). Incubate for 1 hr at RT.

hFAB Rhodamine Housekeeping Antibodies |6

5. Wash: 6 x 5 min at RT with TBST.
6. Image on the ChemiDoc™ MP Imaging Systemwith Image Lab™ Touch Software. Configure a
multichannel imaging protocol using the Rhodamine
option under Application > Blots for the hFAB
Rhodamine Antibody and the StarBright B700
option under Application > Blots for the StarbrightBlue 700 Secondary Antibody (for Image Lab TouchSoftware, version 2.0 and later software releases).

hFAB Rhodamine Housekeeping Antibodies |7

Ordering Information
Catalog #Description
hFAB Rhodamine Housekeeping Antibodies
12004163
Anti-Actin hFAB Rhodamine Antibody, 200 µl
12004164
Anti-Actin hFAB Rhodamine Antibody, 40 µl
12004165
Anti-Tubulin hFAB Rhodamine Antibody, 200 µl
12004166
Anti-Tubulin hFAB Rhodamine Antibody, 40 µl
12004167
Anti-GAPDH hFAB Rhodamine Antibody, 200 µl
12004168
Anti-GAPDH hFAB Rhodamine Antibody, 40 µl
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Bio-Rad Laboratories, Inc.
2000 Alfred Nobel Drive, Hercules, CA 94547 USA
510-741-1000
10000068702 Ver B (12001956)

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