HydA2 | Iron-hydrogenase HydA2

AS09 600 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Chlamydomonas reinhardtii

Product Information

Immunogen

Recombinant, full length Chlamydomonas reinhardtii HydA-2 UniProt: Q8VZZ0

Host Rabbit

Clonality Polyclonal

Purity Serum

Format Lyophilized

Quantity 100 µl

Reconstitution For reconstitution add 100 µl of sterile water

Storage Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.

Tested applications Western blot (WB)

Recommended dilution 1 : 5000 (WB)

Expected | apparent MW 53,7 | 47,3 kDa (after a signal peptide is cleaved)

Reactivity

Confirmed reactivity Chlamydomonas reinhardtii, recombinant HydA2 expressed in E.coli

Predicted reactivity

Ostreococcus sp.

Species of your interest not listed? Contact us

Not reactive in No confirmed exceptions from predicted reactivity are currently known

Application examples

Application example

western blot detection using anti-HydA antibodies

50 ng of purified protein (HydA1 and HydA2) were separated on 10% SDS-PAGE and blotted 25 min to PVDF membrane. Filters were blocked 1h with 3% low-fat milk powder in PBS-T (0.1% TWEEN 20) and probed with anti-HydA2 (AS09 600, 1:5000, over night at 4°C) and secondary anti-rabbit (1:10 000, 1 h) antibody (HRP conjugated, manufacture Pierce) in PBS-T containing 3% low fat milk powder. Antibody incubations were followed by washings in PBS-T (10, +10min and PBS (+5, +5 min). All washing steps were performed at RT with agitation. Signal was detected with chemiluminescent detection, using CCD camer. Exposure time was 20 min.

Courtesy Dr. Thomas Happe, Ruhr-Universität Bochum, Germany

Additional information

In Chlamydomonas HydA is present in low levels of 1 µg/liter of culture. Therefore, an induction of cells by anaerobic adaptation or sulfur depravation (10 x higher amount than with anaerobic adaptation) is necessary for successful detection using this antibody. Methods of HydA induction are described in Hemschemeier et al. 2009.

To detect HydA in Chlamydomonas extracts amount loaded per well corresponds to 2 µg of chlorophyll for sulfur deprived cells, where relatively much HydA1 is synthesized or corresponds to 2-4 µg of artificially anaerobic induced cultures, where the HydA1 protein level is lower.

This antibody is recognizing 1 ng of recombinant HydA protein.

HydA2 (505aa) has a calculated MW of 53.7 kDa, but this is including the signal peptide, which gets cleaved off. The protein without TP can only be estimated, since the cleavage site is known only from in silico analysis. It has a calculated MW of 47.3 kDa and should run in the gel also according to its size.

HydA1 (497aa) has a calculated MW of 53.1 kDa, but this is including the signal peptide, which gets cleaved off. The protein without TP has a calculated MW of 47.5 kDa and runs according to its size at about 48 kDa

Background

Iron-hydrogenase HydA2 is catalyzing reversible oxidation of molecular hydrogen. Chlamydomonas the protein is present in low levels of 1 µg/liter of culture. Synonymnes: HYD2

Product citations

Picture (footer):	Application example 50 ng of purified protein (HydA1 and HydA2) were separated on 10% SDS-PAGE and blotted 25 min to PVDF membrane. Filters were blocked 1h with 3% low-fat milk powder in PBS-T (0.1% TWEEN 20) and probed with anti-HydA2 (AS09 600, 1:5000, over night at 4°C) and secondary anti-rabbit (1:10 000, 1 h) antibody (HRP conjugated, manufacture Pierce) in PBS-T containing 3% low fat milk powder. Antibody incubations were followed by washings in PBS-T (10, +10min and PBS (+5, +5 min). All washing steps were performed at RT with agitation. Signal was detected with chemiluminescent detection, using CCD camer. Exposure time was 20 min. Courtesy Dr. Thomas Happe, Ruhr-Universität Bochum, Germany
background:	Iron-hydrogenase HydA2 is catalyzing reversible oxidation of molecular hydrogen. Chlamydomonas the protein is present in low levels of 1 µg/liter of culture. Synonymnes: HYD2
calculated \| apparent molecular mass [kDa]:	53,7 \| 47,3 kDa (after a signal peptide is cleaved)
Clonality:	Polyclonal
Format:	Lyophilized
Host:	Rabbit
immunogen:	Recombinant, full length Chlamydomonas reinhardtii HydA-2 UniProt: Q8VZZ0
Purity:	Serum
Quantity:	100 µl
recommended dilution:	1 : 5000 (WB)
Reconstitution:	For reconstitution add 100 µl of sterile water
storage:	Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.
tested applications:	Western blot (WB)
Confirmed reactivity:	Chlamydomonas reinhardtii, recombinant HydA2 expressed in E.coli
predicted reactivity:	Ostreococcus sp. Species of your interest not listed? Contact us
not reactive in:	No confirmed exceptions from predicted reactivity are currently known
additional information:	In Chlamydomonas HydA is present in low levels of 1 µg/liter of culture. Therefore, an induction of cells by anaerobic adaptation or sulfur depravation (10 x higher amount than with anaerobic adaptation) is necessary for successful detection using this antibody. Methods of HydA induction are described in Hemschemeier et al. 2009. To detect HydA in Chlamydomonas extracts amount loaded per well corresponds to 2 µg of chlorophyll for sulfur deprived cells, where relatively much HydA1 is synthesized or corresponds to 2-4 µg of artificially anaerobic induced cultures, where the HydA1 protein level is lower. This antibody is recognizing 1 ng of recombinant HydA protein.
additional information (application):	HydA2 (505aa) has a calculated MW of 53.7 kDa, but this is including the signal peptide, which gets cleaved off. The protein without TP can only be estimated, since the cleavage site is known only from in silico analysis. It has a calculated MW of 47.3 kDa and should run in the gel also according to its size. HydA1 (497aa) has a calculated MW of 53.1 kDa, but this is including the signal peptide, which gets cleaved off. The protein without TP has a calculated MW of 47.5 kDa and runs according to its size at about 48 kDa